LAGE-1 tumor associated nucleic acids

ABSTRACT

The invention describes the LAGE-1 tumor associated gene, including fragments, allelic variants and splice variants thereof. Also included are polypeptides and fragments thereof encoded by such genes, and antibodies relating thereto. Methods and products also are provided for diagnosing and treating conditions characterized by expression of a LAGE-1 gene product.

RELATED APPLICATIONS

This application is a divisional of U.S. patent application Ser. No.09/341,829, filed Oct. 18, 1999, now issued as U.S. Pat. No. 6,794,131,which is a national stage filing under 35 U.S.C. § 371 of PCTInternational application PCT/US98/01445, filed Jan. 27, 1998, andpublished under PCT Article 21(2) in English, which is a continuation-inpart of and claims priority under 35 U.S.C. § 120 from U.S. patentapplication Ser. No. 08/791,495, now abandoned, filed Jan. 27, 1997,each of which is incorporated by reference herein in its entirety.

FIELD OF THE INVENTION

This invention relates to nucleic acid molecules and encodedpolypeptides which are expressed preferentially in tumors. The nucleicacid molecules and encoded polypeptides are useful in, inter alia,diagnostic and therapeutic contexts.

BACKGROUND OF THE INVENTION

The phenotypic changes which distinguish a tumor cell from its normalcounterpart are often the result of one or more changes to the genome ofthe cell. The genes which are expressed in tumor cells, but not innormal counterparts, can be termed “tumor associated” genes. These tumorassociated genes are markers for the tumor phenotype. The expression oftumor associated genes can also be an essential event in the process oftumorigenesis.

Typically, the host recognizes as foreign the tumor associated geneswhich are not expressed in normal non-tumorigenic cells. Thus, theexpression of tumor associated genes can provoke an immune responseagainst the tumor cells by the host. Tumor associated genes can also beexpressed in normal cells within certain tissues without provoking animmune response. In such tissues, expression of the gene and/orpresentation of an ordinarily immunologically recognizable fragment ofthe protein product on the cell surface may not provoke an immuneresponse because the immune system does not “see” the cells inside theseimmunologically privileged tissues. Examples of immunologicallyprivileged tissues include brain and testis

The discovery of tumor associated expression of a gene provides a meansof identifying a cell as a tumor cell. Diagnostic compounds can be basedon the tumor associated gene, and used to determine the presence andlocation of tumor cells. Further, when the tumor associated gene isessential for an aspect of the tumor phenotype (e.g., unregulated growthor metastasis), the tumor associated gene can be used to providetherapeutics such as antisense nucleic acids which can reduce orsubstantially eliminate expression of that gene, thereby reducing orsubstantially eliminating the phenotypic aspect which depends on theexpression of the particular tumor associated gene.

As previously noted, the polypeptide products of tumor associated genescan be the targets for host immune surveillance and provoke selectionand expansion of one or more clones of cytotoxic T lymphocytes specificfor the tumor associated gene product. Examples of this phenomenoninclude proteins and fragments thereof encoded by the MAGE family ofgenes, the tyrosinase gene, the Melan-A gene, the BAGE gene, the GAGEgene, the RAGE family of genes, the PRAME gene and the brain glycogenphosphorylase gene, as are detailed below. Thus, tumor associatedexpression of genes suggests that such genes can encode proteins whichwill be recognized by the immune system as foreign and thus provide atarget for tumor rejection. Such genes encode “tumor rejection antigenprecursors”, or TRAPs, which may be used to generate therapeutics forenhancement of the immune system response to tumors expressing suchgenes and proteins.

The process by which the mammalian immune system recognizes and reactsto foreign or alien materials is a complex one. An important facet ofthe system is the T cell response. This response requires that T cellsrecognize and interact with complexes of cell surface molecules,referred to as human leukocyte antigens (“HLA”), or majorhistocompatibility complexes (“MHCs”), and peptides. The peptides arederived from larger molecules which are processed by the cells whichalso present the HLA/MHC molecule. See in this regard Male et al.,Advanced Immunology (J.P. Lipincott Company, 1987), especially chapters6-10. The interaction of T cells and complexes of HLA/peptide isrestricted, requiring a T cell specific for a particular combination ofan HLA molecule and a peptide. If a specific T cell is not present,there is no T cell response even if its partner complex is present.Similarly, there is no response if the specific complex is absent, butthe T cell is present. The mechanism is involved in the immune system'sresponse to foreign materials, in autoimmune pathologies, and inresponses to cellular abnormalities. Much work has focused on themechanisms by which proteins are processed into the HLA bindingpeptides. See, in this regard, Barinaga, Science 257: 880, 1992; Fremontet al., Science 257: 919, 1992; Matsumura et al., Science 257: 927,1992; Latron et al., Science 257: 964, 1992.

The mechanism by which T cells recognize cellular abnormalities has alsobeen implicated in cancer. For example, in PCT applicationPCT/US92/04354, filed May 22, 1992, published on Nov. 26, 1992, andincorporated by reference, a family of genes is disclosed, which areprocessed into peptides which, in turn, are expressed on cell surfaces,which can lead to lysis of the tumor cells by specific CTLs. The genesare said to code for “tumor rejection antigen precursors” or “TRAP”molecules, and the peptides derived therefrom are referred to as “tumorrejection antigens” or “TRAs”. See Traversari et al., J. Exp. Med.176:1453-1457, 1992; van der Bruggen et al., Science 254: 1643,1991; DePlaen et al., Immunogenetics 40:360-369, 1994 for further information onthis family of genes. Also, see U.S. patent application Ser. No.807,043, filed Dec. 12, 1991, now U.S. Pat. No. 5,342,774.

In U.S. patent application Ser. No. 938,334, now U.S. Pat. No.5,405,940, the disclosure of which is incorporated by reference,nonapeptides are taught which are presented by the HLA-A1 molecule. Thereference teaches that given the known specificity of particularpeptides for particular HLA molecules, one should expect a particularpeptide to bind one HLA molecule, but not others. This is important,because different individuals possess different HLA phenotypes. As aresult, while identification of a particular peptide as being a partnerfor a specific HLA molecule has diagnostic and therapeuticramifications, these are only relevant for individuals with thatparticular HLA phenotype. There is a need for further work in the area,because cellular abnormalities are not restricted to one particular HLAphenotype, and targeted therapy requires some knowledge of the phenotypeof the abnormal cells at issue.

In U.S. patent application Ser. No. 008,446, filed Jan. 22, 1993 andincorporated by reference, the fact that the MAGE-1 expression productis processed to a second TRA is disclosed. This second TRA is presentedby HLA-Cw16 molecules, also known as HLA-C*1601. The disclosure showsthat a given TRAP can yield a plurality of TRAs.

In U.S. patent application Ser. No. 994,928, filed Dec. 22, 1992, andincorporated by reference herein, tyrosinase is described as a tumorrejection antigen precursor. This reference discloses that a moleculewhich is produced by some normal cells (e.g., melanocytes), is processedin tumor cells to yield a tumor rejection antigen that is presented byHLA-A2 molecules.

In U.S. patent application Ser. No. 08/032,978, filed Mar. 18, 1993, andincorporated herein by reference in its entirety, a second TRA, notderived from tyrosinase is taught to be presented by HLA-A2 molecules.The TRA is derived from a TRAP, but is coded for by a known MAGE gene.This disclosure shows that a particular HLA molecule may present TRAsderived from different sources.

In U.S. patent application Ser. No. 079,110, filed Jun. 17, 1993 andentitled “Isolated Nucleic Acid Molecules Coding For BAGE TumorRejection Antigen Precursors” and Ser. No. 196,630, filed Feb. 15, 1994,and entitled “Isolated Peptides Which form Complexes with MHC MoleculeHLA-C-Clone 10 and Uses Thereof” the entire disclosures of which areincorporated herein by reference, an unrelated tumor rejection antigenprecursor, the so-called “BAGE” precursor, is described. TRAs arederived from the TRAP and also are described. They form complexes withMHC molecule HLA-C-Clone 10.

In U.S. patent application Ser. No. 096,039, filed Jul. 22, 1993 andentitled “Isolated Nucleic Acid Molecules Coding for GAGE TumorRejection Antigen Precursors” and Ser. No. 250,162, filed May 27, 1994and entitled “Method for Diagnosing a Disorder by Determining Expressionof GAGE Tumor Rejection Antigen Precursors”, the entire disclosures ofwhich are incorporated herein by reference, another unrelated tumorrejection antigen precursor, the so-called “GAGE” precursor, isdescribed. The GAGE precursor is not related to the BAGE or the MAGEfamily.

In U.S. patent application Ser. No. 08/408,015, filed Mar. 21, 1995, andentitled “RAGE Tumor Rejection Antigen Precursors”, incorporated hereinby reference in its entirety, another TRAP is taught which is notderived from any of the foregoing genes. The TRAP is referred to asRAGE. In U.S. patent application Ser. No. 08/530,015, filed Sep. 20,1995, and entitled “Isolated RAGE-1 Derived Peptides Which Complex withHLA-B7 Molecules and Uses Thereof”, also incorporated by reference, theTRA derived form one member of the RAGE family of genes is taught to bepresented by HLA-B7 molecules. This disclosure shows that additionalTRAPs and TRAs can be derived from different sources.

In U.S. patent application Ser. No. 08/253,503, filed Jun. 3, 1994, andentitled “Isolated Nucleic Acid Molecule Which Codes for a TumorRejection Antigen Precursor Which is Processed to an Antigen Presentedby HLA-B44”, incorporated herein by reference in its entirety, anotherTRAP is taught which is not derived from any of the foregoing genes. Thegene encoding the TRAP is referred to as MUM-1. A tumor rejectionantigen, LB-33B, is described in the application.

In U.S. patent application Ser. No. 08/373,636, filed Jan. 17, 1995, andentitled “Isolated Nucleic Acid Molecule Which Codes for a TumorRejection Antigen Precursor Which is Processed to Antigens Presented byHLA Molecules and Uses Thereof”, incorporated herein by reference in itsentirety, other TRAPs are taught which are derived from LB33 andpresented by HLA-B13, HLA-Cw6, HLA-A28 and HLA-A24.

In PCT publication WO96/10577, published Apr. 11, 1996, and entitled“Isolated Nucleic Acid Molecule Coding for a Tumor Rejection AntigenPrecursor DAGE and Uses Thereof”, incorporated herein by reference inits entirety, another TRAP is taught which is not derived from any ofthe foregoing genes. The TRAP was referred to as DAGE, but is nowreferred to as PRAME. A tumor rejection antigen is described in theapplication which is presented by HLA-A24.

In U.S. patent application Ser. No. 08/487,135, filed Jun. 7, 1995, andentitled “Isolated Nucleic Acid Molecule, Peptides Which Form Complexeswith MHC Molecule HLA-A2 and Uses Thereof”, incorporated herein byreference in its entirety, another TRAP is taught which is not derivedfrom any of the foregoing genes. The TRAP is referred to as NAG. VariousTRAs derived from NAG and presented by HLA-A2 are taught in thisapplication.

In U.S. patent application Ser. No. 08/403,388, filed Mar. 14, 1995, andentitled “Isolated Nucleic Acid Molecules Which Are Members of theMAGE-Xp Family and Uses Thereof”, incorporated herein by reference inits entirety, three TRAPs are taught which are not derived from any ofthe foregoing genes. These TRAPs are referred to as MAGE-Xp2, MAGE-Xp3and MAGE-Xp4.

The work which is presented by the papers, patents and patentapplications described above deal, for the most part, with the MAGEfamily of genes, the BAGE gene, the GAGE gene and the RAGE family ofgenes. It now has been discovered that additional genes similarly areexpressed in a tumor associated pattern.

The invention is elaborated upon further in the disclosure whichfollows.

SUMMARY OF THE INVENTION

The genes which are believed to encode tumor rejection antigenprecursors were referred to originally as LL-1 tumor associated genes(LL-1.1 and LL-1.2). One of the two genes, originally termed LL-1.2, isnow known as NY-ESO-1 as described in U.S. patent application Ser. No.08/725,182. The other LL-1 gene, LL-1.1, is now known as LAGE-1 and doesnot show homology to the MAGE family of genes, to the BAGE gene, theGAGE gene, the RAGE family of genes, the LB33/MUM-1 gene, the PRAMEgene, the NAG gene or the MAGE-Xp family of genes. Thus the inventionrelates to the LAGE-1 gene expressed specifically in certain tumorcells, tumor rejection antigen precursors encoded by the LAGE-1 gene, aswell as related molecules and applications of these various entities.

The invention provides isolated nucleic acid molecules, unique fragmentsof those molecules, expression vectors containing the foregoing, andhost cells transfected with those molecules. The invention also providesisolated polypeptides and agents which bind such polypeptides, includingantibodies. Kits for detecting the presence of a LAGE-1 tumor associatedpolypeptide precursor additionally are provided. The foregoing can beused in the diagnosis or treatment of conditions characterized by theexpression of a LAGE-1 tumor-specific polypeptide or precursor thereof.

According to one aspect of the invention, an isolated nucleic acidmolecule is provided. The molecule hybridizes under stringent conditionsto a molecule having a nucleotide sequence selected from the groupconsisting of the nucleotide sequence of SEQ ID NO:4 and the nucleotidesequence of SEQ ID NO:6. The isolated nucleic acid molecule is a LAGE-1tumor associated polypeptide precursor and codes for a LAGE-1 tumorassociated polypeptide, including allelic variants of LAGE-1 tumorassociated polypeptides. The invention further embraces nucleic acidmolecules that differ from the foregoing isolated nucleic acid moleculesin codon sequence to the degeneracy of the genetic code. The inventionalso embraces complements of the foregoing nucleic acids.

In preferred embodiments, the isolated nucleic acid molecule comprises amolecule selected from the group consisting of the nucleic acid sequenceof SEQ ID NO:4 and the nucleic acid sequence of SEQ ID NO:6. Morepreferably, the isolated nucleic acid molecule comprises a moleculeselected from the group consisting of the coding region of the nucleicacid sequence of SEQ ID. NO:4 and the coding region of the nucleic acidsequence of SEQ ID NO:6.

According to another aspect of the invention, an isolated nucleic acidmolecule is provided which comprises a molecule selected from the groupconsisting of a unique fragment of nucleotides 1-993 of SEQ ID NO:4between 12 and 992 nucleotides in length, a unique fragment ofnucleotides 1-746 of SEQ ID NO:6 between 12 and 745 nucleotides inlength, and complements thereof. The unique fragments exclude nucleicacid molecules which consist only of fragments of SEQ ID NO:8 andfragments of SEQ ID NO:8 having 5 or fewer contiguous nucleotides of SEQID NO:4 OR SEQ ID NO:6. In preferred embodiments, the unique fragment isat least 14, 15, 16, 17, 18, 20 or 22 contiguous nucleotides ofnucleotides 1-993 of SEQ ID NO:4, nucleotides 1-746 SEQ ID NO:6, orcomplements thereof. In another embodiment, the isolated nucleic acidmolecule consists of between 12 and 32 contiguous nucleotides ofnucleotides 1-993 of SEQ ID NO:4, nucleotides 1-746 of SEQ ID NO:6, orcomplements of such nucleic acid molecules.

According to another aspect of the invention, the invention involvesexpression vectors, and host cells transformed or transfected with suchexpression vectors, comprising the nucleic acid molecules describedabove. The expression vectors and/or host cells preferably include anucleic acid molecule which codes for a HLA molecule. Of course, anHLA-encoding nucleic acid molecule can also be contained in a separateexpression vector.

According to another aspect of the invention, an isolated polypeptideencoded by a nucleic acid molecule which hybridizes under stringentconditions to a molecule selected from the group consisting of thenucleic acid sequence of SEQ ID NO:4 and the nucleic acid sequence ofSEQ ID NO:6, nucleic acid molecules which vary from the foregoingaccording to the degeneracy of the genetic code, complements and allelicvariants of any of the foregoing nucleic acid molecules. Preferredpolypeptides are those which include the amino acid sequence of SEQ IDNO:5, the amino acid sequence of SEQ ID NO:7, the amino acid sequence ofSEQ ID NO:5 or SEQ ID NO:7 having a glutamine to arginine substitutionat residue 6, the amino acid sequence of SEQ ID NO:5 or SEQ ID NO:7having a glutamine to glutamic acid substitution at residue 89, and theamino acid sequence of SEQ ID NO:5 having an arginine to tryptophansubstitution at residue 138.

In another aspect of the invention, isolated LAGE-1 polypeptides whichinclude amino acids 89-93 of SEQ ID NO:5 or 7 are provided. Preferredembodiments of such polypeptides include isolated LAGE-1 polypeptideswhich include amino acids 71-93, 71-98, 89-98, 89-111, or 71-111 of SEQID NO:5 or 7. Nucleic acids which encode such polypeptides also areprovided.

According to another aspect of the invention, isolated LAGE-1bpolypeptides which include amino acids 142-148 of SEQ ID NO:5, aminoacids 187-205 of SEQ ID NO:5, or amino acids 164-179 of SEQ ID NO:5 areprovided. Preferably such isolated polypeptides include amino acids134-210 of SEQ ID NO:5. Isolated nucleic acids which encode suchpolypeptides also are provided.

According to yet another aspect of the invention, an isolatedpolypeptide is provided which comprises a molecule selected from thegroup consisting of a unique fragment of SEQ ID NO:5 between 9 and 209amino acids in length and a unique fragment of SEQ ID NO:7 between 9 and179 amino acids in length. The unique fragment is not a polypeptideconsisting of fragments of SEQ ID NO:9. Preferably, the unique fragmentof the isolated polypeptide binds to a polypeptide-binding agent. Inpreferred embodiments, the polypeptide-binding agent is an antibody or acytotoxic T lymphocyte.

The invention also provides isolated polypeptides which selectively binda LAGE-1 protein or fragments thereof. Isolated binding polypeptidesinclude antibodies and fragments of antibodies (e.g., Fab, F(ab)₂, Fdand antibody fragments which include a CDR III region which bindsselectively to the LAGE-1 proteins of the invention). The isolatedbinding polypeptides include monoclonal antibodies.

The invention in another aspect involves a kit for detecting thepresence of the expression of a LAGE-1 tumor associated polypeptideprecursor. Such kits employ two or more of the above-described moleculesisolated in separate containers and packaged in a single package. In onesuch kit, a pair of isolated nucleic acid molecules is provided, each ofthe pair consisting essentially of a molecule selected from the groupconsisting of a 12-32 nucleotide contiguous segment of SEQ ID NO:4 andcomplements thereof, and a 12-32 nucleotide contiguous segment of SEQ IDNO:6 and complements thereof, wherein the contiguous segments arenonoverlapping. Preferably, the pair of isolated nucleic acid moleculesis constructed and arranged to selectively amplify an isolated nucleicacid molecule which hybridizes under stringent conditions to a moleculeselected from the group consisting of the nucleic acid sequence of SEQID NO:4, the nucleic acid sequence of SEQ ID NO:6, nucleic acidmolecules which differ from the above in codon sequence due to thedegeneracy of the genetic code, complements and allelic variantsthereof. In certain embodiments, the pair of isolated nucleic acidmolecules is PCR primers. Preferably one of the primers is a contiguoussegment of SEQ ID NO:4 and another of the primers is a complement ofanother contiguous segment of SEQ ID NO:4. In other preferredembodiments, one of the primers is a contiguous segment of SEQ ID NO:6and another of the primers is the complement of another contiguoussegment of SEQ ID NO:6.

According to still another aspect of the invention, a method fordiagnosing a disorder characterized by the expression of a LAGE-1nucleic acid molecule or an expression product thereof is provided. Themethod involves contacting a biological sample isolated from a subjectwith an agent that selectively binds a LAGE-1 nucleic acid molecule oran expression product thereof. In certain embodiments, the nucleic acidmolecule hybridizes under stringent conditions to a molecule selectedfrom the group consisting of the nucleic acid sequence of SEQ ID NO:4and the nucleic acid sequence of SEQ ID NO:6, and which codes for atumor associated polypeptide. In other embodiments, the agent is abinding agent which selectively binds to a LAGE-1 tumor associatedpolypeptide, such as an antibody, cytotoxic T lymphocyte, polypeptide,and the like. The method further involves determining the interactionbetween the agent and the nucleic acid molecule or expression productthereof as a determination of the disorder. In preferred embodiments,the agent is a DNA molecule comprising SEQ ID NO:4 or SEQ ID NO:6, or aunique fragment thereof. In certain embodiments, the interaction betweenthe agent and the nucleic acid molecule is determined by amplifying atleast a portion of the nucleic acid molecule.

According to another aspect of the invention, a method for treating asubject with a disorder characterized by expression of an LAGE-1 tumorassociated polypeptide is provided. The method involves administering tothe subject an amount of an agent, which agent enriches selectively inthe subject the presence of complexes of a HLA molecule and a tumorrejection antigen which is derived from a LAGE-1 tumor associatedpolypeptide coded for by one of the foregoing nucleic acid molecules.The amount of the agent administered is sufficient to ameliorate thedisorder. Preferably the agent is a LAGE-1 polypeptide, or animmunogenic fragment thereof.

According to yet another aspect of the invention, a method for treatinga subject with a disorder characterized by expression of a LAGE-1nucleic acid molecule or an expression product thereof is provided. Themethod includes administering to the subject an amount of autologouscytolytic T cells sufficient to ameliorate the disorder, wherein thecytolytic T cells are specific for complexes of an HLA molecule and aLAGE-1 tumor associated polypeptide or an immunogenic fragment thereof.

In another aspect, the invention provides methods for treating a subjectwith a disorder characterized by expression of a LAGE-1 nucleic acidmolecule or an expression product thereof. The methods includeadministering to the subject an amount of a LAGE-1 tumor associatedpolypeptide or an immunogenic fragment thereof sufficient to amelioratethe disorder.

According to another aspect of the invention, methods for enrichingselectively a population of T cells with cytolytic T cells specific fora LAGE-1 tumor associated polypeptide are provided. The methods includecontacting an isolated population of T cells with an agent presenting acomplex of a LAGE-1 tumor associated polypeptide or an immunogenicfragment thereof and a HLA presenting molecule in an amount sufficientto selectively enrich the isolated population of T cells with thecytolytic T cells. Preferably the agent is a cell which expresses aLAGE-1 tumor associated polypeptide and a HLA molecule. In certainpreferred embodiments, the LAGE-1 tumor associated polypeptide isencoded by a nucleic acid molecule having the nucleotide sequence of SEQID NO:4 or SEQ ID NO:6.

According to other aspects of the invention, vaccine compositions whichinclude a nucleic acid encoding at least one LAGE-1 epitope, a LAGE-1polypeptide and/or a cell which expresses LAGE-1 nucleic acid orpolypeptide, or immunogenic fragments thereof, are provided. The vaccinecompositions are useful for increasing an immune response in a subject.

Use of the foregoing compositions in the preparation of medicaments isalso provided. In particular, use of the composition in the preparationof a medicament for treating cancer is provided.

These and other objects of the invention will be described in furtherdetail in connection with the detailed description of the invention.

BRIEF DESCRIPTION OF THE FIGURES

FIG. 1 depicts the nucleotide sequences of LL-1 clones 2, 3 and 4 (nowknown as LAGE-1 clone 2 [LAGE-1b], NY-ESO-1 and LAGE-1 clone 4[LAGE-1a], respectively).

BRIEF DESCRIPTION OF THE SEQUENCES

SEQ ID NO:1 is the nucleotide sequence of LAGE-1 clone 1.

SEQ ID NO:2 is the nucleotide sequence of primer SL25.

SEQ ID NO:3 is the nucleotide sequence of primer BLE56.

SEQ ID NO:4 is the nucleotide sequence of LAGE-1 clone 2, also known asLAGE-1b.

SEQ ID NO:5 is the amino acid sequence of the translation product ofLAGE-1 clone 2.

SEQ ID NO:6 is the nucleotide sequence of LAGE-1 clone 4, also known asLAGE-1a.

SEQ ID NO:7 is the amino acid sequence of the translation product ofLAGE-1 clone 4.

SEQ ID NO:8 is the nucleotide sequence of NY-ESO-1, formerly known asLL-1.2 clone 3.

SEQ ID NO:9 is the amino acid sequence of the translation product ofNY-ESO-1, formerly known as LL-1.2 clone 3.

SEQ ID NO:10 is the nucleotide sequence of primer BLE70.

SEQ ID NO:11 is the nucleotide sequence of primer BLE71.

SEQ ID NO:12 is the nucleotide sequence of primer BLE72.

SEQ ID NO:13 is the nucleotide sequence of primer BLE73.

SEQ ID NO:14 is the nucleotide sequence of primer BLE74.

DETAILED DESCRIPTION OF THE INVENTION

The examples which follow show the isolation of nucleic acid moleculeswhich code for polypeptides and are expressed preferentially in tumorsamples and tumor-derived cell lines. These isolated nucleic acidmolecules, however, are not homologous with any of the previouslydisclosed coding sequences described in the references set forth supra.Hence, one aspect of the invention is an isolated nucleic acid moleculewhich includes all or a unique portion of the nucleotide sequence setforth in SEQ ID NO:4 or SEQ ID NO:6. These sequences are not MAGE, BAGE,GAGE, RAGE, LB33/MUM-1, PRAME, NAG, MAGE-Xp or NY-ESO-1 sequences, aswill be seen by comparing them to the sequence of any of the genesdescribed in the references.

The invention thus involves LAGE-1 nucleic acids, polypeptides encodedby those nucleic acids, functional modifications and variants of theforegoing, useful fragments of the foregoing, as well as therapeuticsand diagnostics related thereto.

Also a part of the invention are those nucleic acid sequences which alsocode for a LAGE-1 tumor associated polypeptide and which hybridize to anucleic acid molecule consisting of the nucleotide sequence set forth inSEQ ID NO:4 (LAGE-1b) or SEQ ID NO:6 (LAGE-1a), under stringentconditions, but which are not nucleic acid molecules consisting of thenucleotide sequence set forth in SEQ ID NO:8 (NY-ESO-1). LAGE-1 nucleicacids are characterized by at least 90% identity with exon 2 of LAGE-1.Preferably the nucleic acid identity with exon 2 of LAGE-1 is at least95% and most preferably is at least 99%. Complements of the foregoingare also embraced by the invention.

Other criteria for establishing that a nucleic acid is part of theLAGE-1 family are shown in FIG. 1. For example, in addition to thenucleotide sequence of the coding region of the LAGE-1 gene andexpression products thereof, the length and composition of intronspresent in the LAGE-1 gene can be used to establish the relation of anucleic acid to LAGE-1. Sequences upstream and downstream of the codingregion also are useful for distinguishing LAGE-1 nucleic acids fromnon-LAGE-1 nucleic acids. The iso-electric point of the encoded proteinsor fragments of the encoded proteins also can serve as distinguishingcharacteristics of nucleic acids related to LAGE-1.

Such nucleic acids are termed tumor associated polypeptide precursors,and may be DNA, RNA, or composed of mixed deoxyribonucleotides andribonucleotides. The tumor associated polypeptide precursors can alsoincorporate synthetic non-natural nucleotides. A tumor associatednucleic acid or polypeptide is a nucleic acid or polypeptide expressedpreferentially in tumor cells. Various methods for determining theexpression of a nucleic acid and/or a polypeptide in normal and tumorcells are known to those of skill in the art and are described furtherbelow. As used herein, tumor associated polypeptides include proteins,protein fragments, and peptides. In particular, tumor associatedpolypeptides include TRAPs and TRAs.

The term “stringent conditions” as used herein refers to parameters withwhich the art is familiar. More specifically, stringent conditions, asused herein, refers to hybridization at 65° C. in hybridization buffer(3.5×SSC, 0.02% Ficoll, 0.02% polyvinyl pyrolidone, 0.02% Bovine SerumAlbumin, 25 mM NaH₂PO₄ (pH 7), 0.5% SDS, 2 mM EDTA). SSC is 0.15M sodiumchloride/0.15M sodium citrate, pH 7; SDS is sodium dodecyl sulphate; andEDTA is ethylenediaminetetracetic acid. After hybridization, themembrane upon which the nucleic acid is transferred is washed at 2×SSCat room temperature and then at 0.1×SSC/0.1×SDS at 65° C. SSC is 0.15Msodium chloride/0.15M sodium citrate, pH 7; SDS is sodium dodecylsulphate; and EDTA is ethylenediamine tetraacetic acid.

There are other conditions, reagents, and so forth which can be used,which result in the same degree of stringency (see, e.g. MolecularCloning: A Laboratory Manual, J. Sambrook, et al., eds., Second Edition,Cold Spring Harbor Laboratory Press, Cold Spring Harbor, New York, 1989,or Current Protocols in Molecular Biology, F. M. Ausubel, et al., eds.,John Wiley & Sons, Inc., New York). The skilled artisan will be familiarwith such conditions, and thus they are not given here. It will beunderstood, however, that the skilled artisan will be able to manipulatethe conditions in a manner to permit the clear identification ofhomologs and alleles of LAGE-1 nucleic acid molecules of the invention.The skilled artisan also is familiar with the methodology for screeningcells, preferably cancer cells, and libraries for expression of suchmolecules which then are routinely isolated, followed by isolation ofthe pertinent nucleic acid and sequencing.

The nucleic acids disclosed herein are useful for determining theexpression of LAGE-1 according to standard hybridization procedures. Thenucleic acids also can be used to express LAGE-1 polypeptides in vitroor in vivo. The nucleic acids also can be used to prepare fragments ofLAGE-1 polypeptides useful for e.g., preparation of antibodies. Manyother uses will be apparent to the skilled artisan.

In screening for LAGE-1 family members, a Southern blot may be performedusing the foregoing conditions, together with a radioactive probe.Preferably hybridizations are performed using probes comprising LAGE-1exon2 and/or intron 2, or portions thereof. After washing the membraneto which the nucleic acid is finally transferred, the membrane can beplaced against x-ray film to detect the radioactive signal.

The invention also includes degenerate nucleic acids which includealternative codons to those present in the native materials. Forexample, serine residues are encoded by the codons TCA, AGT, TCC, TCG,TCT and AGC. Each of the six codons is equivalent for the purposes ofencoding a serine residue. Thus, it will be apparent to one of ordinaryskill in the art that any of the serine-encoding nucleotide triplets maybe employed to direct the protein synthesis apparatus, in vitro or invivo, to incorporate a serine residue. Similarly, nucleotide sequencetriplets which encode other amino acid residues include, but are notlimited to: CCA, CCC, CCG and CCT (proline codons); CGA, CGC, CGG, CGT,AGA and AGG (arginine codons); ACA, ACC, ACG and ACT (threonine codons);AAC and AAT (asparagine codons); and ATA, ATC and ATT (isoleucinecodons). Other amino acid residues may be encoded similarly by multiplenucleotide sequences. Thus, the invention embraces degenerate nucleicacids that differ from the biologically isolated nucleic acids in codonsequence due to the degeneracy of the genetic code.

The invention also provides isolated unique fragments of SEQ ID NO:4 orSEQ ID NO:6, or complements of SEQ ID NO:4 or SEQ ID NO:6. A uniquefragment is one that is a ‘signature’ for the larger nucleic acid. It,for example, is long enough to assure that its precise sequence is notfound in molecules outside of the LAGE-1 family as defined by claim 1.In particular, a unique fragment of LAGE-1 is one which would nothybridize under stringent conditions to a nucleic acid molecule havingthe nucleotide sequence of SEQ ID NO:8. Unique fragments can be used asprobes in Southern blot assays to identify family members or can be usedin amplification assays such as those employing PCR. As known to thoseskilled in the art, large probes such as 200 nucleotides or more (e.g.,200, 250, 300, 400, 500 nucleotides) are preferred for certain uses suchas Southern blots, while smaller fragments will be preferred for usessuch as PCR. Unique fragments also can be used to produce fusionproteins for generating antibodies or for generating immunoassaycomponents. Unique fragments further can be used as antisense moleculesto inhibit the expression of the LAGE-1 proteins of the invention,particularly for therapeutic purposes as described in greater detailbelow.

As will be recognized by those skilled in the art, the size of theunique fragment will depend upon its conservancy in the genetic code.Thus, some regions of SEQ ID NO:4 or SEQ ID NO:6, and their complements,will require longer segments to be unique while others will require onlyshort segments, typically between 12 and 32 nucleotides (e.g. 12, 13,14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31and 32 nucleotides long). Virtually any segment of SEQ ID NO:4 or SEQ IDNO:6, or their complements, that is 18 or more nucleotides in lengthwill be unique. Unique fragments of LAGE-1, however, exclude fragmentscompletely composed of the nucleotide sequence of SEQ ID NO:8 (encodingthe NY-ESO-1 polypeptide) which overlaps SEQ ID NO:4 or SEQ ID NO:6. Afragment which is completely composed of the sequence of SEQ ID NO:8 isone which does not include any of the nucleotides unique to LAGE-1. Incertain embodiments, unique fragments of LAGE-1 include at least 5contiguous nucleotides which are not present in SEQ ID NO:8 (e.g. whichare present in SEQ ID NO:4 or SEQ ID NO:6); preferred unique fragmentsare those which have 6, 7, 8, 9, 10, 12, 14, 16, 18, 20, or morenucleotides which are not present in SEQ ID NO:8. Thus a moleculeconsisting of a fragment of SEQ ID NO:8 with 4 nucleotides of SEQ IDNO:4 or SEQ ID NO:6 added on the 5′ or 3′ end is not a unique fragment.Those skilled in the art are well versed in methods for selecting suchsequences, typically on the basis of the ability of the unique fragmentto selectively distinguish the sequence of interest from non-familymembers. A comparison of the sequence of the LAGE-1 fragment to theNY-ESO-1 nucleic acid sequence and to other sequences deposited in knowndata bases typically is all that is necessary, although in vitroconfirmatory hybridization and sequencing analysis may be performed.

For any pair of PCR primers constructed and arranged to selectivelyamplify, for example, a LAGE-1 nucleic acid, a LAGE-1 specific primermay be used. Such a primer is a contiguous stretch of LAGE-1 whichhybridizes selectively to LAGE-1 and not other nucleic acids. Such aspecific primer would fully hybridize to a contiguous stretch ofnucleotides only in LAGE-1, but would hybridize at most only in part togenes that do not share the nucleotides to which the LAGE-1 specificprimer binds. For efficient PCR priming and LAGE-1 identification, theLAGE-1 specific primer should be constructed and arranged so it does nothybridize efficiently at its 3′ end to genes other than LAGE-1. Thekinetics of hybridization then will strongly favor hybridization at the5′ end. In this instance, 3′ initiated PCR extension will occur onlywhen both the 5′ and 3′ ends hybridize to the nucleic acid. Primers forselective amplification of LAGE-1 clone 2 and/or LAGE-1 clone 4 can beselected from portions of LAGE-1 which share lesser homology withNY-ESO-1 (see FIG. 1; compare SEQ ID NO:4, SEQ ID NO:6 and SEQ ID NO:8).In such cases, the LAGE-1 specific primers can be designed to prime DNAsynthesis on either strand of the LAGE-1 gene, described herein as theantisense or the sense strands. Preferably the area of non-identity isat least one to four nucleotides in length and forms the 3′ end of theLAGE-1 specific primer. Such a primer would be perfectly complementaryand contiguous with its complement in LAGE-1. The 3′ end of the primerwould hybridize to its complement in the antisense strand and initiateextension. In genes other than LAGE-1, the lack of nucleotide sequenceidentity would substantially eliminate hybridization of the 3′ end ofthe LAGF, 1 specific primer to the antisense strand 5′ of the insert.The mismatch generated at the 3′ end of the primer when hybridized togenes other than LAGE-1 would preclude efficient amplification of thosegenes. Exemplary primers include BLE72 (SEQ ID NO:12) which spansnucleotides 265-283 of SEQ ID NO:6. Other primers which containnucleotide sequences not found in NY-ESO-1 can be prepared by one ofskill in the art by comparison of the sequences of SEQ ID NO:4 or SEQ IDNO:6 with SEQ ID NO:8. Portions of SEQ ID NO:6 identical to SEQ ID NO:4would serve equally well as LAGE-1 specific primers. Other exemplaryprimers can differ from the above by addition or deletion of 1, 2, 3, 4,5, or more nucleotides from the 5′ end of the primer.

Similarly, one of ordinary skill in the art can select primers from thenucleotide sequence of SEQ ID NO:8 for selective amplification ofNY-ESO-1 mRNA sequences. For example, exemplary primers specific forNY-ESO-1 include BLE73 (SEQ ID NO:13), and BLE74 (SEQ ID NO:14) which isa sense primer located in SEQ ID NO:8 at nucleotides 262-281 (homologousto the position of BLE72 in SEQ ID NO:6, e.g., nucleotides 264-283). Asdemonstrated in the Examples below, primer pairs specific to LAGE-1 orNY-ESO-1 can be used to distinguish the expression of the genes in cellsand tissues. Other exemplary primers can differ from the above byaddition or deletion of 1, 2, 3, 4, 5, or more nucleotides from the 5′end of the primers above. One of ordinary skill in the art can determinewith no more than routine experimentation the preferred primers forselective amplification of particular LAGE-1 clones.

In certain cases, the primers chosen to distinguish the LAGE-1 clonesprovide amplified products which are readily distinguishable bymolecular size. For example, LAGE-1 primers can be chosen which initiateextension on opposite sides of the splice site by hybridizing tosequences which are identical or nearly so and which hybridize 5′ of the5′ splice site and 3′ of the 3′ splice site. Because LAGE-1 clone 2 mRNAcontains a portion of the gene which is spliced out in formation ofLAGE-1 clone 4 mRNA (see FIG. 1; i.e., nucleotides 469-697 of SEQ IDNO:4), amplification products derived from LAGE-1 clone 2 using suchprimers will be longer than amplification products derived from LAGE-1clone 4 (by about 229 base pairs). This difference may be distinguishedreadily using standard methods in the art including agarose andacrylamide gel electrophoresis.

Additional methods which can distinguish nucleotide sequences ofsubstantial homology, such as ligase chain reaction (“LCR”) and othermethods, will be apparent to skilled artisans.

As used herein with respect to nucleic acids, the term “isolated” means:(i) amplified in vitro by, for example, polymerase chain reaction (PCR);(ii) recombinantly produced by cloning; (iii) purified, as by cleavageand gel separation; or (iv) synthesized by, for example, chemicalsynthesis. An isolated nucleic acid is one which is readily manipulableby recombinant DNA techniques well known in the art. Thus, a nucleotidesequence contained in a vector in which 5′ and 3′ restriction sites areknown or for which polymerase chain reaction (PCR) primer sequences havebeen disclosed is considered isolated but a nucleic acid sequenceexisting in its native state in its natural host is not. An isolatednucleic acid may be substantially purified, but need not be. Forexample, a nucleic acid that is isolated within a cloning or expressionvector is not pure in that it may comprise only a tiny percentage of thematerial in the cell in which it resides. Such a nucleic acid isisolated, however, as the term is used herein because it is readilymanipulable by standard techniques known to those of ordinary skill inthe art.

The invention also provides isolated polypeptides which include uniquefragments of SEQ ID NO:5 or SEQ ID NO:7. Such polypeptides are useful,for example, alone or as fusion proteins to generate antibodies, as acomponents of an immunoassay, or for determining the LAGE-1 proteinbinding specificity of HLA molecules and/or CTL clones. The term“isolated”, as used herein in reference to a polypeptide, means apolypeptide encoded by an isolated nucleic acid sequence, as well aspolypeptides synthesized by, for example, chemical synthetic methods,and polypeptides separated from biological materials, and then purifiedusing conventional protein analytical or preparatory procedures. ThusLAGE-1 proteins include polypeptides having amino acids 89-93 of SEQ IDNO:5 or 7. Preferred embodiments of such polypeptides include LAGE-1polypeptides which include amino acids 71-93, 71-98, 89-98, 89-111, or71-111 of SEQ ID NO:5 or 7. The foregoing amino acid sequences are notfound in the NY-ESO-1 protein. Nucleic acids which encode the foregoingpolypeptides also are embraced by the invention. LAGE-1b polypeptideswhich include amino acids encoded by the alternatively spliced intron 2of LAGE-1 also are provided. These polypeptides in certain embodimentscontain amino acids 142-148 of SEQ ID NO:5, amino acids 187-205 of SEQID NO:5, or amino acids 164-179 of SEQ ID NO:5. Preferably suchpolypeptides include amino acids 134-210 of SEQ ID NO:5. These aminoacid sequences are not found in the NY-ESO-1 or LAGE-1aproteins. Nucleicacids which encode such LAGE-1b polypeptides also are embraced by theinvention. Preferably, a LAGE-1 protein is at least 90%, more preferably95%, and most preferably 99% identical to the amino acid sequence setforth in either SEQ ID NO:5 or SEQ ID NO:7.

A unique fragment of an LAGE-1 protein, in general, has the features andcharacteristics of unique fragments as discussed above in connectionwith nucleic acids. Thus a protein fragment which consists only of aportion of SEQ ID NO:9 is not a unique fragment of LAGE-1. As will berecognized by those skilled in the art, the size of the unique fragmentwill depend upon factors such as whether the fragment constitutes aportion of a conserved protein domain. Thus, some regions of SEQ ID NO:5and SEQ ID NO:7, will require longer segments to be unique while otherswill require only short segments, typically between 5 and 12 amino acids(e.g. 5, 6, 7, 8, 9, 10, 11 and 12 amino acids long). Virtually anysegment of SEQ ID NO:5 or SEQ ID NO:7 which excludes SEQ ID NO:9, thatis 10 or more amino acids in length will be unique. Preferably, uniquefragments of LAGE-1 include at least 2, more preferably 3 and mostpreferably 5 contiguous amino acids which are not present in SEQ ID NO:9(e.g. which are present in SEQ ID NO:5 or SEQ ID NO:7).

Unique fragments of a polypeptide preferably are those fragments whichretain a distinct functional capability of the polypeptide. Functionalcapabilities which can be retained in a unique fragment of a polypeptideinclude interaction with antibodies, interaction with other polypeptidesor fragments thereof, selective binding of nucleic acids, and enzymaticactivity. A tumor rejection antigen is an example of a unique fragmentof a tumor associated polypeptide which retains the functionalcapability of HLA binding and interaction with cytotoxic T lymphocytes.Tumor rejection antigens presented by HLA class I molecules typicallyare 9 amino acids in length, although peptides of 8, 9 and 10 and moreamino acids also retain the capability to interact with HLA andcytotoxic T lymphocyte to an extent effective to provoke a cytotoxic Tlymphocyte response (see, e.g., Van den Eynde & Brichard, Curr. Opin.Immunol. 7:674-681, 1995; Coulie et al., Stem Cells 13:393-403, 1995).

Those skilled in the art are well versed in methods for selecting uniqueamino acid sequences, typically on the basis of the ability of theunique fragment to selectively distinguish the sequence of interest fromnon-LAGE-1 family polypeptides, particularly NY-ESO-1. A comparison ofthe sequence of the fragment to those on known data bases typically isall that is necessary. Certain functional aspects of unique fragments ofthe LAGE-1 polypeptides can be determined by employing well-knowncomputer algorithms to compare LAGE-1 fragments to fragments of otherpolypeptides. For example, an HLA-peptide binding algorithm (availableon the National Institutes of Health website [bimas.dcrt.nih.gov];Parker et al., J. Immunol. 152:163, 1994) can be used to distinguish theHLA binding properties of LAGE-1 peptides from those of NY-ESO-1peptides. For example, the HLA-B7 binding score (half time ofdissociation) of a LAGE-1 peptide containing amino acids 114-123 of SEQID NO:7 is predicted to be 30-fold greater than a NY-ESO-1 peptidehaving the corresponding amino acids (114-123) of SEQ ID NO:9.

The skilled artisan will also realize that conservative amino acidsubstitutions may be made in LAGE-1 polypeptides to provide functionallyactive homologs of the foregoing polypeptides, i.e, the homologs retainthe functional capabilities of the LAGE-1 polypeptides. As used herein,a “conservative amino acid substitution” refers to an amino acidsubstitution which does not alter the relative charge or sizecharacteristics of the protein in which the amino acid substitution ismade. Conservative substitutions of amino acids include substitutionsmade amongst amino acids within the following groups: (a) M, I, L, V;(b) F, Y, W; (c) K, R, H; (d) A, G; (e) S, T; (f) Q, N; and (g) E, D.

Functionally equivalent variants of LAGE-1 polypeptides, i.e., variantsof LAGE-1 polypeptides which retain the function of the natural LAGE-1polypeptides, can be prepared according to methods for alteringpolypeptide sequence known to one of ordinary skill in the art such asare found in references which compile such methods, e.g. MolecularCloning: A Laboratory Manual, J. Sambrook, et al., eds., Second Edition,Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y., 1989, orCurrent Protocols in Molecular Biology, F. M. Ausubel, et al., eds.,John Wiley & Sons, Inc., New York. Exemplary functionally equivalentvariants of the LAGE-1 polypeptides include conservative amino acidsubstitutions of SEQ ID NO:5 and SEQ ID NO:7. Conservative amino-acidsubstitutions in the amino acid sequence of LAGE-1 polypeptides toproduce functionally equivalent variants of LAGE-1 polypeptidestypically are made by alteration of the nucleic acid encoding LAGE-1polypeptides (SEQ ID NO:4, SEQ ID NO:6). Such substitutions can be madeby a variety of methods known to one of ordinary skill in the art. Forexample, amino acid substitutions may be made by PCR-directed mutation,site-directed mutagenesis according to the method of Kunkel (Kunkel,Proc. Nat. Acad. Sci. US.A. 82: 488-492, 1985), or by chemical synthesisof a gene encoding a LAGE-1 polypeptide. Where amino acid substitutionsare made to a small unique fragment of a LAGE-1 polypeptide, such as a 9amino acid peptide, the substitutions can be made by directlysynthesizing the peptide. The activity of functionally equivalentfragments of LAGE-1 polypeptides can be tested by cloning the geneencoding the altered LAGE-1 polypeptide into a bacterial or mammalianexpression vector, introducing the vector into an appropriate host cell,expressing the altered LAGE-1 polypeptide, and testing for a functionalcapability of the LAGE-1 polypeptides as disclosed herein.

As mentioned above, the invention embraces antisense oligonucleotidesthat selectively bind to a nucleic acid molecule encoding an LAGE-1protein, to decrease transcription and/or translation of LAGE-1 genes.This is desirable in virtually any medical condition wherein a reductionin LAGE-1 gene product expression is desirable, including to reduce anyaspect of a tumor cell phenotype attributable to LAGE-1 gene expression.Antisense molecules, in this manner, can be used to slow down or arrestsuch aspects of a tumor cell phenotype.

As used herein, the term “antisense oligonucleotide” or “antisense”describes an oligonucleotide that is an oligoribonucleotide,oligodeoxyribonucleotide, modified oligoribonucleotide, or modifiedoligodeoxyribonucleotide which hybridizes under physiological conditionsto DNA comprising a particular gene or to an mRNA transcript of thatgene and, thereby, inhibits the transcription of that gene and/or thetranslation of that mRNA. The antisense molecules are designed so as tointerfere with transcription or translation of a target gene uponhybridization with the target gene. Those skilled in the art willrecognize that the exact length of the antisense oligonucleotide and itsdegree of complementarity with its target will depend upon the specifictarget selected, including the sequence of the target and the particularbases which comprise that sequence. It is preferred that the antisenseoligonucleotide be constructed and arranged so as to bind selectivelywith the target under physiological conditions, i.e., to hybridizesubstantially more to the target sequence than to any other sequence inthe target cell under physiological conditions. Based upon SEQ ID NO:4and/or SEQ ID NO:6, or upon allelic or homologous genomic and/or DNAsequences, one of skill in the art can easily choose and synthesize anyof a number of appropriate antisense molecules for use in accordancewith the present invention. In order to be sufficiently selective andpotent for inhibition, such antisense oligonucleotides should compriseat least 7 (Wagner et al., Nature Biotechnology 14:840-844, 1996) and,more preferably, at least 15 consecutive bases which are complementaryto the target. Most preferably, the antisense oligonucleotides comprisea complementary sequence of 20-30 bases. Although oligonucleotides maybe chosen which are antisense to any region of the gene or mRNAtranscripts, in preferred embodiments the antisense oligonucleotidescorrespond to N-terminal or 5′ upstream sites such as translationinitiation, transcription initiation or promoter sites. In addition,3′-untranslated regions may be targeted. Targeting to mRNA splicingsites has also been used in the art but may be less preferred ifalternative mRNA splicing occurs. In addition, the antisense istargeted, preferably, to sites in which mRNA secondary structure is notexpected (see, e.g., Sainio et al., Cell Mol. Neurobiol. 14(5):439-457,1994) and at which proteins are not expected to bind. Finally, although,SEQ ID Nos:4 and 6 disclose cDNA sequences, one of ordinary skill in theart may easily derive the genomic DNA corresponding to the cDNAs of SEQID Nos:4 and 6. Thus, the present invention also provides for antisenseoligonucleotides which are complementary to the genomic DNAcorresponding to SEQ ID Nos:4 and 6. Similarly, antisense to allelic orhomologous DNAs and genomic DNAs are enabled without undueexperimentation.

In one set of embodiments, the antisense oligonucleotides of theinvention may be composed of “natural” deoxyribonucleotides,ribonucleotides, or any combination thereof. That is, the 5′ end of onenative nucleotide and the 3′ end of another native nucleotide may becovalently linked, as in natural systems, via a phosphodiesterinternucleoside linkage. These oligonucleotides may be prepared by artrecognized methods which may be carried out manually or by an automatedsynthesizer. They also may be produced recombinantly by vectors.

In preferred embodiments, however, the antisense oligonucleotides of theinvention also may include “modified” oligonucleotides. That is, theoligonucleotides may be modified in a number of ways which do notprevent them from hybridizing to their target but which enhance theirstability or targeting or which otherwise enhance their therapeuticeffectiveness.

The term “modified oligonucleotide” as used herein describes anoligonucleotide in which (1) at least two of its nucleotides arecovalently linked via a synthetic internucleoside linkage (i.e., alinkage other than a phosphodiester linkage between the 5′ end of onenucleotide and the 3′ end of another nucleotide) and/or (2) a chemicalgroup not normally associated with nucleic acids has been covalentlyattached to the oligonucleotide. Preferred synthetic internucleosidelinkages are phosphorothioates, alkylphosphonates, phosphorodithioates,phosphate esters, alkylphosphonothioates, phosphoramidates, carbamates,carbonates, phosphate triesters, acetamidates, peptides, andcarboxymethyl esters.

The term “modified oligonucleotide” also encompasses oligonucleotideswith a covalently modified base and/or sugar. For example, modifiedoligonucleotides include oligonucleotides having backbone sugars whichare covalently attached to low molecular weight organic groups otherthan a hydroxyl group at the 3′ position and other than a phosphategroup at the 5′ position. Thus modified oligonucleotides may include a2′-O-alkylated ribose group. In addition, modified oligonucleotides mayinclude sugars such as arabinose instead of ribose. Modifiedoligonucleotides also can include base analogs such as C-5 propynemodified bases (Wagner et al., Nature Biotechnology 14:840-844, 1996).The present invention, thus, contemplates pharmaceutical preparationscontaining modified antisense molecules that are complementary to andhybridizable with, under physiological conditions, nucleic acidsencoding LAGE-1 proteins, together with pharmaceutically acceptablecarriers.

Antisense oligonucleotides may be administered as part of apharmaceutical composition. Such a pharmaceutical composition mayinclude the antisense oligonucleotides in combination with any standardphysiologically and/or pharmaceutically acceptable carriers which areknown in the art. The compositions should be sterile and contain atherapeutically effective amount of the antisense oligonucleotides in aunit of weight or volume suitable for administration to a patient. Theterm “pharmaceutically acceptable” means a non-toxic material that doesnot interfere with the effectiveness of the biological activity of theactive ingredients. The term “physiologically acceptable” refers to anon-toxic material that is compatible with a biological system such as acell, cell culture, tissue, or organism. The characteristics of thecarrier will depend on the route of administration. Physiologically andpharmaceutically acceptable carriers include diluents, fillers, salts,buffers, stabilizers, solubilizers, and other materials which are wellknown in the art.

It will also be seen from the examples that the invention embraces theuse of the LAGE-1 sequences in expression vectors, as well as totransfect host cells and cell lines, be these prokaryotic (e.g., E.coli), or eukaryotic (e.g., CHO cells, COS cells, yeast expressionsystems and recombinant baculovirus expression in insect cells).Especially useful are mammalian cells such as mouse, hamster, pig, goat,primate, etc. They may be of a wide variety of tissue types, includingmast cells, fibroblasts, oocytes and lymphocytes, and they may beprimary cells or cell lines. Specific examples include dendritic cells,U293 cells, peripheral blood leucocytes, bone marrow stem cells andembryonic stem cells. The expression vectors require that the pertinentsequence, i.e., those nucleic acids described supra, be operably linkedto a promoter. As it is believed that a human HLA class I moleculepresents a tumor rejection antigen derived from these genes, theexpression vector may also include a nucleic acid sequence coding forthe HLA molecule that presents any particular tumor rejection antigenderived from these genes and polypeptides. Alternatively, the nucleicacid sequence coding for such a HLA molecule can be contained within aseparate expression vector. In a situation where the vector containsboth coding sequences, the single vector can be used to transfect a cellwhich does not normally express either one. Where the coding sequencesfor the tumor rejection antigen precursor and the HLA molecule whichpresents it are contained on separate expression vectors, the expressionvectors can be cotransfected. The tumor rejection antigen precursorcoding sequence may be used alone, when, e.g. the host cell alreadyexpresses a HLA molecule which presents a LAGE-1 TRA. Of course, thereis no limit on the particular host cell which can be used. As thevectors which contain the two coding sequences may be used in anyantigen-presenting cells if desired, and the gene for tumor rejectionantigen precursor can be used in host cells which do not express a HLAmolecule which presents a LAGE-1 TRA. Further, cell-free transcriptionsystems may be used in lieu of cells.

The skilled artisan can determine which HLA molecule binds to tumorrejection antigens derived from LAGE-1 clone 2 and/or LAGE-1 clone 4tumor rejection antigen precursors by, e.g., experiments utilizingantibodies to block specifically individual HLA class I molecules. Forexample, antibodies which bind selectively to HLA-A2 will preventefficient presentation of TRAs specifically presented by HLA-A2. Thus,if TRAs derived from LAGE-1 are presented by HLA-A2, then the inclusionof anti-HLA-A2 antibodies in an in vitro assay will block thepresentation of the LAGE-1 TRA. An assay for determining the nature ofthe HLA molecule is found in U.S. patent application Ser. No.08/530,569. Briefly, in determining the HLA molecule type, inhibitionexperiments were carried out where the production of tumor necrosisfactor (TNF) by cytotoxic T lymphocyte (CTL) clone 263/17 was tested inthe presence of monoclonal antibodies directed against HLA molecules oragainst CD4/CD8 accessory molecules. Four monoclonal antibodies werefound to inhibit the production of TNF by CTL 263/17: monoclonalantibody W6/32, which is directed against all HLA class I molecules(Parham et al., J. Immunol. 123:342, 1979), antibody B1.23.2 whichrecognizes HLA-B and C molecules (Rebai et al., Tissue Antigens 22:107,1983), antibody ME-1 which specifically recognizes HLA-B7 (Ellis et al.,Hum. Immunol. 5:49, 1982) and antibody B9.4.1 against CD8. No inhibitionwas found with antibodies directed against HLA Class II DR molecules(L243: Lampson et al., J. Immunol. 125:293 , 1980), against HLA-A3 (GAPA3: Berger et al., Hybridoma 1:87, 1982) or against CD4 (13B.8.82). Theconclusion was that CTL 263/17 was of the CD8 type, and recognized anantigen presented by HLA-B7. Similar experiments using widely availableanti-HLA antibodies can be performed to determine the nature of a HLAmolecule.

As used herein, a “vector” may be any of a number of nucleic acids intowhich a desired sequence may be inserted by restriction and ligation fortransport between different genetic environments or for expression in ahost cell. Vectors are typically composed of DNA although RNA vectorsare also available. Vectors include, but are not limited to, plasmidsand phagemids. A cloning vector is one which is able to replicate in ahost cell, and which is further characterized by one or moreendonuclease restriction sites at which the vector may be cut in adeterminable fashion and into which a desired DNA sequence may beligated such that the new recombinant vector retains its ability toreplicate in the host cell. In the case of plasmids, replication of thedesired sequence may occur many times as the plasmid increases in copynumber within the host bacterium or just a single time per host beforethe host reproduces by mitosis. In the case of phage, replication mayoccur actively during a lytic phase or passively during a lysogenicphase. An expression vector is one into which a desired DNA sequence maybe inserted by restriction and ligation such that it is operably joinedto regulatory sequences and may be expressed as an RNA transcript.Vectors may further contain one or more marker sequences suitable foruse in the identification of cells which have or have not beentransformed or transfected with the vector. Markers include, forexample, genes encoding proteins which increase or decrease eitherresistance or sensitivity to antibiotics or other compounds, genes whichencode enzymes whose activities are detectable by standard assays knownin the art (e.g. β-galactosidase or alkaline phosphatase), and geneswhich visibly affect the phenotype of transformed or transfected cells,hosts, colonies or plaques. Preferred vectors are those capable ofautonomous replication and expression of the structural gene productspresent in the DNA segments to which they are operably joined.

As used herein, a coding sequence and regulatory sequences are said tobe “operably” joined when they are covalently linked in such a way as toplace the expression or transcription of the coding sequence under theinfluence or control of the regulatory sequences. If it is desired thatthe coding sequences be translated into a functional protein, two DNAsequences are said to be operably joined if induction of a promoter inthe 5′ regulatory sequences results in the transcription of the codingsequence and if the nature of the linkage between the two DNA sequencesdoes not (1) result in the introduction of a frame-shift mutation, (2)interfere with the ability of the promoter region to direct thetranscription of the coding sequences, or (3) interfere with the abilityof the corresponding RNA transcript to be translated into a protein.Thus, a promoter region would be operably joined to a coding sequence ifthe promoter region were capable of effecting transcription of that DNAsequence such that the resulting transcript might be translated into thedesired protein or polypeptide.

The precise nature of the regulatory sequences needed for geneexpression may vary between species or cell types, but shall in generalinclude, as necessary, 5′ non-transcribing and 5′ non-translatingsequences involved with the initiation of transcription and translationrespectively, such as a TATA box, capping sequence, CAAT sequence, andthe like. Especially, such 5′ non-transcribing regulatory sequences willinclude a promoter region which includes a promoter sequence fortranscriptional control of the operably joined gene. Regulatorysequences may also include enhancer sequences or upstream activatorsequences as desired. The vectors of the invention may optionallyinclude 5′ leader or signal sequences, 5′ or 3′. The choice and designof an appropriate vector is within the ability and discretion of one ofordinary skill in the art.

Expression vectors containing all the necessary elements for expressionare commercially available and known to those skilled in the art. SeeMolecular Cloning: A Laboratory Manual, J. Sambrook, et al., eds.,Second Edition, Cold Spring Harbor Laboratory Press, Cold Spring Harbor,New York, 1989. Cells are genetically engineered by the introductioninto the cells of heterologous DNA (RNA) encoding the LAGE-1 tumorassociated polypeptide or fragment or variant thereof. That heterologousDNA (RNA) is placed under operable control of transcriptional elementsto permit the expression of the heterologous DNA in the host cell.

Preferred systems for mRNA expression in mammalian cells are those suchas pRc/CMV (available from Invitrogen, San Diego, Calif.) that contain aselectable marker such as a gene that confers G418 resistance (whichfacilitates the selection of stably transfected cell lines) and thehuman cytomegalovirus (CMV) enhancer-promoter sequences. Additionally,suitable for expression in primate or canine cell lines is the pCEP4vector (Invitrogen), which contains an Epstein Barr virus (EBV) originof replication, facilitating the maintenance of plasmid as a multicopyextrachromosomal element. Another expression vector is the pEF-BOSplasmid containing the promoter of polypeptide Elongation Factor 1α,which stimulates efficiently transcription in vitro. The plasmid isdescribed by Mishizuma and Nagata (Nuc. Acids Res. 18:5322, 1990), andits use in transfection experiments is disclosed by, for example,Demoulin (Mol. Cell. Biol. 16:4710-4716, 1996). Still another preferredexpression vector is an adenovirus, described by Stratford-Perricaudet,which is defective for E1 and E3 proteins (J. Clin. Invest. 90:626-630,1992). The use of the adenovirus as an Adeno.P1A recombinant isdisclosed by Warnier et al., in intradermal ionjection in mice forimmunization against P1A (Int. J Cancer, 67:303-310, 1996).

The invention also embraces so-called expression kits, which allow theartisan to prepare a desired expression vector or vectors. Suchexpression kits include at least separate portions of each of thepreviously discussed coding sequences. Other components may be added, asdesired, as long as the previously mentioned sequences, which arerequired, are included.

The invention also involves agents which bind to LAGE-1 polypeptides andin certain embodiments preferably to unique fragments of the LAGE-1polypeptides. Such binding partners can be used in screening assays todetect the presence or absence of the LAGE-1 polypeptide and inpurification protocols to isolate LAGE-1 polypeptides. Likewise, suchbinding partners can be used to selectively target drugs, toxins orother molecules to tumor cells which present LAGE-1 tumor associatedpolypeptides. In this manner, tumor cells which express LAGE-1polypeptides can be treated with cytotoxic compounds.

The invention, therefore, involves antibodies or fragments of antibodieshaving the ability to selectively bind to LAGE-1 polypeptides, andpreferably to unique fragments thereof. Antibodies include polyclonaland monoclonal antibodies, prepared according to conventionalmethodology.

Significantly, as is well-known in the art, only a small portion of anantibody molecule, the paratope, is involved in the binding of theantibody to its epitope (see, in general, Clark, W. R. (1986) TheExperimental Foundations of Modern Immunology Wiley & Sons, Inc., NewYork; Roitt, I. (1991) Essential Immunology, 7th Ed., BlackwellScientific Publications, Oxford). The pFc′ and Fc regions, for example,are effectors of the complement cascade but are not involved in antigenbinding. An antibody from which the pFc′ region has been enzymaticallycleaved, or which has been produced without the pFc′ region, designatedan F(ab′)₂ fragment, retains both of the antigen binding sites of anintact antibody. Similarly, an antibody from which the Fc region hasbeen enzymatically cleaved, or which has been produced without the Fcregion, designated an Fab fragment, retains one of the antigen bindingsites of an intact antibody molecule. Proceeding further, Fab fragmentsconsist of a covalently bound antibody light chain and a portion of theantibody heavy chain denoted Fd. The Fd fragments are the majordeterminant of antibody specificity (a single Fd fragment may beassociated with up to ten different light chains without alteringantibody specificity) and Fd fragments retain epitope-binding ability inisolation.

Within the antigen-binding portion of an antibody, as is well-known inthe art, there are complementarity determining regions (CDRs), whichdirectly interact with the epitope of the antigen, and framework regions(FRs), which maintain the tertiary structure of the paratope (see, ingeneral, Clark, 1986; Roitt, 1991). In both the heavy chain Fd fragmentand the light chain of IgG immunoglobulins, there are four frameworkregions (FR1 through FR4) separated respectively by threecomplementarity determining regions (CDR1 through CDR3). The CDRs, andin particular the CDR3 regions, and more particularly the heavy chainCDR3, are largely responsible for antibody specificity.

It is now well-established in the art that the non-CDR regions of amammalian antibody may be replaced with similar regions of conspecificor heterospecific antibodies while retaining the epitopic specificity ofthe original antibody. This is most clearly manifested in thedevelopment and use of “humanized” antibodies in which non-human CDRsare covalently joined to human FR and/or Fc/pFc′ regions to produce afunctional antibody. Thus, for example, PCT International PublicationNumber WO 92/04381 teaches the production and use of humanized murineRSV antibodies in which at least a portion of the murine FR regions havebeen replaced by FR regions of human origin. Such antibodies, includingfragments of intact antibodies with antigen-binding ability, are oftenreferred to as “chimeric” antibodies.

Thus, as will be apparent to one of ordinary skill in the art, thepresent invention also provides for F(ab′)₂, Fab, Fv and Fd fragments;chimeric antibodies in which the Fc and/or FR and/or CDR1 and/or CDR2and/or light chain CDR3 regions have been replaced by homologous humanor non-human sequences; chimeric F(ab′)₂ fragment antibodies in whichthe FR and/or CDR1 and/or CDR2 and/or light chain CDR3 regions have beenreplaced by homologous human or non-human sequences; chimeric Fabfragment antibodies in which the FR and/or CDR1 and/or CDR2 and/or lightchain CDR3 regions have been replaced by homologous human or non-humansequences; and chimeric Fd fragment antibodies in which the FR and/orCDR1 and/or CDR2 regions have been replaced by homologous human ornon-human sequences. The present invention also includes so-calledsingle chain antibodies. Thus, the invention involves polypeptides ofnumerous size and type that bind specifically to LAGE-1 polypeptides.These polypeptides may be derived also from sources other than antibodytechnology. For example, such polypeptide binding agents can be providedby degenerate peptide libraries which can be readily prepared insolution, in immobilized form or as phage display libraries.Combinatorial libraries also can be synthesized of peptides containingone or more amino acids. Libraries further can be synthesized ofpeptoids and non-peptide synthetic moieties.

Phage display can be particularly effective in identifying bindingpeptides useful according to the invention. Briefly, one prepares aphage library (using e.g. m13, fd, or lambda phage), displaying insertsfrom 4 to about 80 amino acid residues using conventional procedures.The inserts may represent a completely degenerate or biased array. Onethen can select phage-bearing inserts which bind to a LAGE-1polypeptide. This process can be is repeated through several cycles ofreselection of phage that bind to the LAGE-1 polypeptide. Repeatedrounds lead to enrichment of phage bearing particular sequences. DNAsequence analysis can be conducted to identify the sequences of theexpressed polypeptides. The minimal linear portion of the sequence thatbinds to the LAGE-1 polypeptide can be determined. One can repeat theprocedure using a biased library containing inserts containing part orall of the minimal linear portion plus one or more additional degenerateresidues upstream or downstream thereof. Thus, the LAGE-1 polypeptidesof the invention can be used to screen peptide libraries, includingphage display libraries, to identify and select peptide binding partnersof the LAGE-1 polypeptides of the invention. Such molecules can be used,as described, for screening assays, for diagnostic assays, forpurification protocols or for targeting drugs, toxins and/or labelingagents (e.g. radioisotopes, fluorescent molecules, etc.) to cells whichpresent LAGE-1 polypeptides on the cell surface. Such binding agentmolecules can also be prepared to bind complexes of an LAGE-1polypeptide and an HLA molecule by selecting the binding agent usingsuch complexes. Drug molecules that would disable or destroy tumor cellswhich express such complexes or LAGE-1 polypeptides are known to thoseskilled in the art and are commercially available. For example, theimmunotoxin art provides examples of toxins which are effective whendelivered to a cell by an antibody or fragment thereof. Examples oftoxins include ribosome-damaging toxins derived from plants or bacterialsuch as ricin, abrin, saporin, Pseudomonas endotoxin, diphtheria toxin,A chain toxins, blocked ricin, etc.

The invention as described herein has a number of uses, some of whichare described herein. First, the invention permits the artisan todiagnose a disorder characterized by expression of the TRAP. Thesemethods involve determining expression of the TRAP gene, and/or TRAsderived therefrom. In the former situation, such determinations can becarried out via any standard nucleic acid determination assay, includingthe polymerase chain reaction as exemplified in the examples below, orassaying with labeled hybridization probes.

The isolation of the TRAP gene also makes it possible to isolate theTRAP molecule itself, especially TRAP molecules containing the aminoacid sequences coded for by SEQ ID NO:4 or SEQ ID NO:6. A variety ofmethodologies well-known to the skilled practitioner can be utilized toobtain isolated TRAP molecules. The protein may be purified from cellswhich naturally produce the protein. Alternatively, an expression vectormay be introduced into cells to cause production of the protein. Inanother method, mRNA transcripts may be microinjected or otherwiseintroduced into cells to cause production of the encoded protein.Translation of mRNA in cell-free extracts such as the reticulocytelysate system also may be used to produce protein. Those skilled in theart also can readily follow known methods for isolating proteins inorder to obtain isolated TRAPs. These include, but are not limited to,immunochromotography, HPLC, size-exclusion chromatography, ion-exchangechromatography and immune-affinity chromatography.

These isolated molecules when processed and presented as the TRA, or ascomplexes of TRA and HLA, such as HLA-A1, HLA-A2, or HLA-B7, may becombined with materials such as adjuvants to produce vaccines useful intreating disorders characterized by expression of the TRAP molecule. Inaddition to LAGE-1 peptides, nucleic acids which encode LAGE peptideepitopes can be used to prepare vaccines. Preparation of nucleic acidsand/or peptides for use in vaccines is well known in the art. When“disorder” is used herein, it refers to any pathological condition wherethe tumor rejection antigen precursor is expressed. An example of such adisorder is cancer, melanoma in particular.

In addition, vaccines can be prepared from cells which present theTRA/HLA complexes on their surface, such as non-proliferative cancercells, non-proliferative transfectants, etcetera. In all cases wherecells are used as a vaccine, these can be cells transfected with codingsequences for one or both of the components necessary to provoke a CTLresponse, or be cells which already express both molecules without theneed for transfection.

Therapeutic approaches based upon the disclosure are premised on aresponse by a subject's immune system, leading to lysis of TRApresenting cells, such as HLA-B7 cells. One such approach is theadministration of autologous CTLs specific to the complex to a subjectwith abnormal cells of the phenotype at issue. It is within the skill ofthe artisan to develop such CTLs in vitro. Generally, a sample of cellstaken from a subject, such as blood cells, are contacted with a cellpresenting the complex and capable of provoking CTLs to proliferate. Thetarget cell can be a transfectant, such as a COS cell of the typedescribed supra. These transfectants present the desired complex oftheir surface and, when combined with a CTL of interest, stimulate itsproliferation. COS cells, such as those used herein are widelyavailable, as are other suitable host cells. Specific production of aCTL is well known to one of ordinary skill in the art. The clonallyexpanded autologous CTLs then are administered to the subject. OtherCTLs specific to LAGE-1 clone 2 and/or LAGE-1 clone 4 may be isolatedand administered by similar methods.

To detail a therapeutic methodology, referred to as adoptive transfer(Greenberg, J. Immunol. 136(5): 1917, 1986; Riddel et al., Science 257:238, 1992; Lynch et al, Eur. J. Immunol. 21: 1403-1410, 1991; Kast etal., Cell 59: 603-614, 1989), cells presenting the desired complex arecombined with CTLs leading to proliferation of the CTLs specificthereto. The proliferated CTLs are then administered to a subject with acellular abnormality which is characterized by certain of the abnormalcells presenting the particular complex. The CTLs then lyse the abnormalcells, thereby achieving the desired therapeutic goal.

The foregoing therapy assumes that at least some of the subject'sabnormal cells present the relevant HLA/TRA complex. This can bedetermined very easily, as the art is very familiar with methods foridentifying cells which present a particular HLA molecule, as well ashow to identify cells expressing DNA of the pertinent sequences, in thiscase a LAGE-1 sequence. Once cells presenting the relevant complex areidentified via the foregoing screening methodology, they can be combinedwith a sample from a patient, where the sample contains CTLs. If thecomplex presenting cells are lysed by the mixed CTL sample, then it canbe assumed that a LAGE-1 derived TRA is being presented, and the subjectis an appropriate candidate for the therapeutic approaches set forthsupra.

Adoptive transfer is not the only form of therapy that is available inaccordance with the invention. CTLs can also be provoked in vivo, usinga number of approaches. One approach is the use of non-proliferativecells expressing the complex. The cells used in this approach may bethose that normally express the complex, such as irradiated tumor cellsor cells transfected with one or both of the genes necessary forpresentation of the complex. Chen et al., Proc. Natl. Acad. Sci. USA 88:110-114 (1991) exemplifies this approach, showing the use of transfectedcells expressing HPV E7 peptides in a therapeutic regime. Various celltypes may be used. Similarly, vectors carrying one or both of the genesof interest may be used. Viral or bacterial vectors are especiallypreferred. For example, nucleic acids which encode a LAGE-1 TRA may beoperably linked to promoter and enhancer sequences which directexpression of the LAGE-1 TRA in certain tissues or cell types. Thenucleic acid may be incorporated into an expression vector. Expressionvectors may be unmodified extrachromosomal nucleic acids, plasmids orviral genomes constructed or modified to enable insertion of exogenousnucleic acids, such as those encoding LAGE-1 TRAs. Nucleic acidsencoding a LAGE-1 TRA also may be inserted into a retroviral genome,thereby facilitating integration of the nucleic acid into the genome ofthe target tissue or cell type. In these systems, the gene of interestis carried by a microorganism, e.g., a Vaccinia virus, retrovirus or thebacteria BCG, and the materials de facto “infect” host cells. The cellswhich result present the complex of interest, and are recognized byautologous CTLs, which then proliferate.

A similar effect can be achieved by combining a TRAP or a stimulatoryfragment thereof with an adjuvant to facilitate incorporation into HLApresenting cells in vivo. The TRAP is processed to yield the peptidepartner of the HLA molecule while the TRA is presented without the needfor further processing. Generally, subjects can receive an intradermalinjection of an effective amount of LAGE-1 TRAP, and/or TRAs derivedtherefrom. Initial doses can be followed by booster doses, followingimmunization protocols standard in the art.

Yet another approach which can be utilized to provoke an immune responseis to provide LAGE-1 TRAs in the form of a nucleic acid encoding aseries of epitopes, known as “polytopes”. The epitopes can be arrangedin sequential or overlapping fashion (see, e.g., Thomson et al., Proc.Natl. Acad. Sci. USA 92:5845-5849, 1995; Gilbert et al., NatureBiotechnol. 15:1280-1284, 1997), with or without the natural flankingsequences, and can be separated by unrelated linker sequences ifdesired. The polytope is processed to generated individual epitopeswhich are recognized by the immune system for generation of immuneresponses.

As part of the immunization protocols, substances which potentiate theimmune response may be administered with nucleic acid or peptidecomponents of a cancer vaccine. Such immune response potentiatingcompound may be classified as either adjuvants or cytokines. Adjuvantsmay enhance the immunological response by providing a reservoir ofantigen (extracellularly or within macrophages), activating macrophagesand stimulating specific sets of lymphocytes. Adjuvants of many kindsare well known in the art; specific examples include MPL (SmithKlineBeecham), a congener obtained after purification and acid hydrolysis ofSalmonella minnesota Re 595 lipopolysaccharide, QS21 (SmithKlineBeecham), a pure QA-21 saponin purified from Quillja saponaria extract,and various water-in-oil emulsions prepared from biodegradable oils suchas squalene and/or tocopherol. Cytokines are also useful in vaccinationprotocols as a result of lymphocyte stimulatory properties. Manycytokines useful for such purposes will be known to one of ordinaryskill in the art, including interleukin-12 (IL-12) which has been shownto enhance the protective effects of vaccines (Science 268: 1432-1434,1995).

When administered, the therapeutic compositions of the present inventionare administered in pharmaceutically acceptable preparations. Suchpreparations may routinely contain pharmaceutically acceptableconcentrations of salt, buffering agents, preservatives, compatiblecarriers, supplementary immune potentiating agents such as adjuvants andcytokines and optionally other therapeutic agents.

The therapeutics of the invention can be administered by anyconventional route, including injection or by gradual infusion overtime. The administration may, for example, be oral, intravenous,intraperitoneal, intramuscular, intracavity, subcutaneous, ortransdermal. When antibodies are used therapeutically, a preferred routeof administration is by pulmonary aerosol. Techniques for preparingaerosol delivery systems containing antibodies are well known to thoseof skill in the art. Generally, such systems should utilize componentswhich will not significantly impair the biological properties of theantibodies, such as the paratope binding capacity (see, for example,Sciarra and Cutie, “Aerosols,” in Remington's Pharmaceutical Sciences,18th edition, 1990, pp 1694-1712). Those of skill in the art can readilydetermine the various parameters and conditions for producing antibodyaerosols without resort to undue experimentation. When using antisensepreparations of the invention, slow intravenous administration ispreferred.

Preparations for parenteral administration include sterile aqueous ornon-aqueous solutions, suspensions, and emulsions. Examples ofnon-aqueous solvents are propylene glycol, polyethylene glycol,vegetable oils such as olive oil, and injectable organic esters such asethyl oleate. Aqueous carriers include water, alcoholic/aqueoussolutions, emulsions or suspensions, including saline and bufferedmedia. Parenteral vehicles include sodium chloride solution, Ringer'sdextrose, dextrose and sodium chloride, lactated Ringer's or fixed oils.Intravenous vehicles include fluid and nutrient replenishers,electrolyte replenishers (such as those based on Ringer's dextrose), andthe like. Preservatives and other additives may also be present such as,for example, antimicrobials, anti-oxidants, chelating agents, and inertgases and the like.

The invention also contemplates gene therapy. The procedure forperforming ex vivo gene therapy is outlined in U.S. Pat. No. 5,399,346and in exhibits submitted in the file history of that patent, all ofwhich are publicly available documents. In general, it involvesintroduction in vitro of a functional copy of a gene into a cell(s) of asubject which contains a defective copy of the gene, and returning thegenetically engineered cell(s) to the subject. The functional copy ofthe gene is under operable control of regulatory elements which permitexpression of the gene in the genetically engineered cell(s). Numeroustransfection and transduction techniques as well as appropriateexpression vectors are well known to those of ordinary skill in the art,some of which are described in PCT application WO95/00654. In vivo genetherapy using vectors such as adenovirus also is contemplated accordingto the invention.

The preparations of the invention are administered in effective amounts.An effective amount is that amount of a pharmaceutical preparation thatalone, or together with further doses, stimulates the desired response.In the case of treating cancer, the desired response is inhibiting theprogression of the cancer. This may involve only slowing the progressionof the disease temporarily, although more preferably, it involveshalting the progression of the disease permanently. This can bemonitored by routine methods or can be monitored according to diagnosticmethods of the invention discussed herein.

Where it is desired to stimulate an immune response using a therapeuticcomposition of the invention, this may involve the stimulation of ahumoral antibody response resulting in an increase in antibody titer inserum, a clonal expansion of cytotoxic lymphocytes, or some otherdesirable immunologic response. It is believed that doses of immunogensranging from one nanogram/kilogram to 100 milligrams/kilogram, dependingupon the mode of administration, would be effective. The preferred rangeis believed to be between 500 nanograms and 500 micrograms per kilogram.The absolute amount will depend upon a variety of factors, including thematerial selected for administration, whether the administration is insingle or multiple doses, and individual patient parameters includingage, physical condition, size, weight, and the stage of the disease.These factors are well known to those of ordinary skill in the art andcan be addressed with no more than routine experimentation.

EXAMPLES Example 1 Isolation of a Sequence Specifically Expressed byMelanoma Cell Line LB373-MEL

Specific cDNA fragments of melanoma cell line LB373-MEL4.0 were enrichedby subtraction of cDNA fragments found in LB243 normal skin cells,according to the representational difference analysis method (RDA)described for DNA by Hubank and Schatz (Nucl. Acids Res. 22: 5640-5648,1994).

Briefly, cellular cDNAs obtained by reverse transcription of poly-A RNAof both LB373-MEL4.0 cells and LB243 normal skin cells primed witholigo-dT were digested by restriction enzyme DpnII. The DpnII fragmentsof cDNAs of each origin (LB373-MEL or LB243 normal skin cells) wereligated with the same set of adapters, divided in several groups andseparately amplified by PCR. The PCR products originating from the samesample were pooled and digested again by DpnII. The DpnII fragments fromthe LB373-MEL cell line (the “tester” cDNA) were ligated with a newadapter set and hybridized with an excess of DpnII DNA fragments derivedfrom the normal skin (the “driver” cDNA). The hybridization mixture wasthen submitted to PCR amplification using the new adaptor set. Onlythose DpnII fragments derived from the tester DNA but not present in thedriver DNA were expected to be amplified exponentially because theycarry primer-complementary sequences at both ends. These tester-specificamplification products were then cloned.

Thirty melanoma-cell specific cDNA clones obtained by this enrichmentprocedure were sequenced and compared with sequences compiled indatabases. Some of the cDNA clones corresponded to known genes withubiquitous expression, some of the cDNA clones corresponded to tumorassociated genes (MAGE-3, MAGE-10, PRAME—formerly known as DAGE) andsome of the cDNA clones were unknown. Among the six unknown clones, onemelanoma-specific cDNA, LAGE-1 clone 1 (SEQ ID NO:1) which was formerlynamed LL-1 clone 1, appeared to have tumor associated expression asdetermined by RT-PCR. The LAGE-1 clone 1 was sequenced and determined tobe 217 base pairs long.

To determine the pattern of expression of LAGE-1, RT-PCR of samples fromvarious tumor and normal tissues was performed. Total RNA of normaltissue and tumor samples was converted to cDNA. An amount of DNAcorresponding to 50 ng of total RNA was then amplified by thirty-twocycles (denature at 94° C. for 60 seconds, anneal at 58° C. for 60 toseconds, and extension at 72° C. for 90 seconds) followed by a finalextension step of 10 minutes at 73° C., using primers SL25 (SEQ ID NO:2)and BLE56 (SEQ ID NO:3), with 0.5U DYNAZYME™ in a buffer, provided bythe supplier (Finnzyme, Finland), containing 10 mM TRIS (pH8.8), 50 mMKCl and 1.5 mM MgCl₂. The total volume of the reaction mixture was 25μl. Ten microliters were separated by electrophoresis on agarose gels. Afragment of the expected size was generated from cDNA of the parentalLB373-MEL cell line and testis. No PCR product was obtained from normalskin cDNA starting materials, nor from a panel of cDNAs from eight othernormal tissues. A faint signal was observed in one normal uterus sample.

Example 2 Isolation of Complete LAGE-1 cDNA Clones from Melanoma CellLine LB373-MEL

A LB373-MEL cDNA library was prepared by reverse transcription of poly-ARNA with an oligo-dT/NotI primer using the Superscript II kit of BRL(Life Technologies, Gaithersburg, Md.). BstXI adapters were ligated tothe ends of the cDNA, and the double stranded cDNA was digested withNotI. These fragments were then cloned into plasmid pCDNA I/Amp digestedwith BstXI and NotI.

To identify full-length LAGE-1 cDNA clones, we used the 137 bp PCRproduct amplified with primers SL25 (SEQ ID NO:2) and BLE56 (SEQ IDNO:3), as a probe to screen 75,000 clones of a cDNA library of LB373-MELcells. The cDNA library was hybridized with the radiolabeled probe andwashed according to standard protocols using 0.4×SSC at 63° C. Thereduced stringency washing conditions were selected to maximizedetection of related cDNA clones. DNA from 25 colonies hybridized to theLAGE-1 probe (0.03% of the total number of colonies), some of which DNAswere isolated and sequenced.

Two clones had sequence identity with the cDNA clone originally isolatedfrom LB373-MEL cells (see Example 1, “clone 1” in FIG. 1). One of theclones contained a sequence that was identical to the 217 base pairs ofLAGE-1 clone 1. This cDNA, referred to as LAGE-1 clone 2 (SEQ ID NO:4,FIG. 1) was determined to be about 993 nucleotides long excluding thepoly A tail. Another hybridizing clone contained a sequence identical tothe last 82 base pairs of LAGE-1 clone 1. This second cDNA, referred toas clone 3 (SEQ ID NO:8, FIG. 1), was determined to be about 744nucleotides in length excluding the poly A tail. Clone 3 is now known tobe NY-ESO-1 (see Chen et al., Proc. Natl. Acad Sci. USA.94(5):1914-1918, 1997) The sequences of clone 2 and clone 3 were 94%identical. Most of the differences in nucleotide sequence between thetwo clones were located in the central region of the cDNAs. A thirdhybridizing clone, clone 4 (SEQ ID NO:6, FIG. 1), was determined to beabout 746 nucleotides in length excluding the poly A tail. Analysis ofthe genomic fragment corresponding to LAGE-1 indicated that the regionencompassing nucleotides 469-697 of clone 2 in FIG. 1 is an intron whichwas not spliced out during the formation of the LAGE-1 clone 2 mRNA.

In all clones, the longest open reading frame (ORF) is believed to beginat the same first ATG in a good transcription initiating context(gccATGc) according to Kozak (J. Biol. Chem. 266: 19867-19870, 1991).The size of the product of translation of completely spliced sequencesof clone 4 (SEQ ID NO:6) and clone 3 (SEQ ID NO:8) derived from genesLAGE-1 and NY-ESO-1 respectively are believed to be in good agreementwith the corresponding ORF (about 19-20 kD for 180 amino acids). Thesequence of the putative protein encoded by NY-ESO-1 is referred to asSEQ ID NO:9. The translation product of partially spliced messenger RNAs(clone 2) from LAGE-1 has an apparent mass of about 25 kDa as determinedby SDS-PAGE analysis of protein prepared by in vitro translation.

Example 3 Expression of LAGE-1 Genes in Normal Tissue and Tumor Samples

To determine the tissue specificity of expression of both LAGE-1 andNY-ESO-1 clones by PCR, we used two primers which correspond tosequences where LAGE-1 and NY-ESO-1 are identical. Primers BLE70 (SEQ IDNO:10) and BLE71 (SEQ ID NO:11), encompassing the main ORF, repeatedlyprovided two signals of nearly 600 base pairs and 850 base pairs whichcorrespond to the fragment sizes of 614 and 842 base pairs expected forthe spliced and unspliced LAGE-1 and NY-ESO-1 cDNAs.

A. Normal Tissues

RT-PCR with primers BLE70 and BLE71 was performed as described inExample 1, except that 20 cycles (denature at 95° C. for 30 seconds,anneal at 60° C. for 1 minute and extension at 70° C. for 3 minutes)were performed followed by 10 cycles with an extension time of 10minutes at 70° C. and 15 minutes at 72° C. for the final extension. Inaddition, the buffer used was 50 mM Tris-HCl, pH9.2 (25° C.), 16 mM(NH₄)₂SO₄, 2.25 mM MgCl₂, 2% (v/v) DMSO and 0.1 % (v/v) TWEEN™ 20(buffer 3 from Expand Long template of Boehringer). Analysis ofamplification products was performed by agarose gel electrophoresis of10 μl of the 25 μl total volume. Samples of various tissue origins weretested again with primers BLE71 and either BLE72 (SEQ ID NO:12, specificfor LAGE-1) or BLE73 (SEQ ID NO:13, specific for NY-ESO-1), for 30cycles with an annealing step at 62° C. for 1 minute and an extensionstep at 72° C. for 2 minutes using 0.5U Dynazyme according tomanufacturer's instructions. The LAGE-1 PCR products were 399 and 628bp, and the NY-ESO-1 product was 274 bp. Since the 628 bp productsystematically appeared in shorter PCR product (spliced), only thelatter is reported in Table I The results are indicated in Table I, withincreasing numbers of plus signs indicating higher levels of RNAexpression in a particular tissue. Samples which did not yield PCRamplification products were retested by using the residual 15 μl ofnegative PCR reactions in a reamplification reaction of 5 or 6 cycles.The results of any reamplification reactions are reported as − or (±) inTable I.

No signal was observed on amplification of genomic DNA using standardPCR conditions with primers BLE70 and BLE71 after 30 and 33 cycles withan annealing step of 60° C. Of the normal tissues analyzed, only thetestis, breast, term placenta, and one out of two uterus samples werepositive (Table I). Investigating the expression of LAGE-1 and NY-ESO-1genes, it was observed that one of the uterus samples expressed a lowlevel of LAGE-1 mRNA, but remained clearly negative for NY-ESO-1 mRNA.Moreover, the seven endometrium and two myometrium RNA samples remainednegative for both genes. On the other hand, both testis samples showedLAGE-1 and NY-ESO-1 expression at a level similar to expression inLB373-MEL4.0 cells. Control amplifications were performed using β-actinspecific primers. All of these cDNA samples strongly expressed β-actinas judged by the signal obtained after a PCR amplification for 21cycles. From a panel of 6 other samples of normal tissues already typednegative with primers SL25 and BLE56, all were also found negative usingLAGE-1 specific BLE72-BLE71 primers. Four of these samples, however,were found positive for NY-ESO-1 expression, but below the threshold of1% of the expression found in LB373-MEL4.0 cells. Those normal sampleswhich exhibited a low level of NY-ESO-1 expression are the skin, thelung, adrenals and breast (Table I).

TABLE I Expression of genes LAGE-1 & NY-ESO-1 in normal tissues (RT-PCR)Tissue LAGE-1 or NY- Sample code NY-ESO-1 LAGE-1 ESO-1 brain JNO10 − − −retina SH8-5 − − − PBL LB33, LB569, LB678 − − − skin LB243 − − (±)breast LB520, LB673 ± − ± heart LB1266 − − − muscle CLO84033 − − lungLB175, LB264 − − ± bone marrow LB214, LB1765 − − liver LB898 − − −kidney BA4, BA25 − − − adrenal LB535, LB538 − − ± glands testis HM31,LB882 ++/+++ ++/+++ ++/+++ prostate HM88, CLO64038 − − − ovary LB1266,CLO84036 − − − term LB692, LB695 ± ± ± placenta uterus LB1022 ± ± −uterus CLO64029^(#) − − endometrium LB1872, LB1874 − − − d.2, 13endometrium 5 samples − − d.19-32 myometrium LB1031, LB1032 − − −^(#)CLO64029: pool of 10 uterus from women (age 15-74) deceased fromtrauma, purchased from Clontech.B. Tumor Samples

Total RNA of tumor samples of the origins indicated in Table II was usedin RT-PCR reactions with LAGE-1 and NY-ESO-1 specific primers (BLE70 andBLE71) as described for normal tissues. Positive samples were retestedby amplification using primers BLE71 and either BLE72 (specific forLAGE-1 transcript) or BLE73 (specific for NY-ESO-1 transcripts) for 30cycles as described above with an annealing step at 62° C. for oneminute and an extension step at 72° C. for 2 minutes using 0.5UDYNAZYME™ according to the manufacturer's instructions. The amount ofRNA and efficiency of cDNA synthesis were controlled for by parallel PCRreactions with a set of mactin specific primers.

Since two normal uterus samples showed a basal expression of geneLAGE-1, tumor samples derived from this organ were studied further.Surprisingly, among 8 tumors tested (4 tumors of the cervix and 4 tumorsof the myometrium), none appear to be positive for LAGE-1 or NY-ESO-1with primers BLE70-BLE71 after 30 and 33 cycles, in spite of a confirmedgood β-actin expression (Table II). Expression of LAGE-1 in uterus wasfurther verified using a commercial RNA sample, derived from uterinetissues from 10 normal women deceased by trauma, which tested negativefor both genes in spite of a good actin expression.

As indicated in Table II, no expression of LAGE-1 or NY-ESO-1 genes wasdetected in colon, kidney, thyroid and brain cancers, nor in leukemias,as assessed by RT-PCR with primers BLE70-BLE71. Expression of LAGE-1 orNY-ESO-1 genes in breast cancer was not rare, but was faint. Theexpression in melanomas, SCLC, sarcomas, head and neck, prostate andbladder tumors was stronger, relatively more frequent and correlatedwith the expression of other genes known to encode antigenic peptides asshown in Table III. The same cDNA samples were tested for expression ofa panel of TRAPs including MAGE-1, -2, -3, -4, -6 and -12, BAGE, PRAME,GAGE-1&2, -3, -4, -5 and -6, and RAGE. A correlation of the expressionof LAGE-1 or NY-ESO-1 with activation of MAGE-1 or other TRAPS is givenin Table III. Half of the samples that were positive for LAGE-1 werealso positive for MAGE-A1 whereas only twelve percent of the negativesamples expressed MAGE-A1. A similar correlation of expression was foundbetween LAGE-1 and MAGE-A3.

Because the expression of LAGE-1 was clearly correlated with that of theMAGE genes, which are activated in tumors upon demethylation of thepromoter region, studies were carried out to determine if demethlyationcould also induce LAGE-1 expression. Phytohemagglutinin-stimulatedlymphoblastoid cells or tumor cells that were negative for LAGE-1 andNY-ESO-1 turned out to express these genes after treatment bydeoxyazacytidine, indicating that methylation is involved in the controlof both genes. In confirmation, it was observed that HpaII sites locatedin exon 1 and in the promoter of LAGE-1 were methylated in bloodmononucleated cells and in tumor cell lines that did not express LAGE-1, whereas the HpaII sites were demethylated in tumor cell lines thatexpressed LAGE-1

LAGE-1 and NY-ESO-1 each accounted for 75% of positive tumor samples.Thus, as demonstrated in Table II both LAGE-1 and NY-ESO-1 family geneswere expressed independently of each other. Sarcomas of varioushistological types preferentially expressed high levels of NY-ESO-1 RNA,independent of the expression of known tumor associated antigensencoding genes.

Using the cDNAs of eight samples which express LAGE-1 and NY-ESO-1 genessimultaneously or NY-ESO-1 alone as templates for RT-PCR reactions,NY-ESO-1 sequence was amplified with primers BLE73 and BLE71 for use asstarting material in sequencing reactions. Primer BLE56 (SEQ ID NO:3)was used to prime sequencing reactions. A unique sequence that wasidentical to the corresponding region of clone 3 derived fromLB373-MEL4.0 cells was observed, demonstrating both the specificity ofthe PCR reactions and the absence of polymorphism in this region of theNY-ESO-1 gene (see FIG. 1).

TABLE II Expression of genes LAGE-1 & NY-ESO-1 in tumors (RT-PCR) LAGE-1& NY- ESO-1 Positive LAGE-1 NY-ESO-1 Sample Number BLE70-BLE71 (%)BLE72-BLE71 BLE73-BLE71 COLON 9 0 ND ND LEUKEMIA 17 0  0% ND NDB-LYMPHOMA 6 1 1 1 MELANOMA 21 7 33% 6 5 HEAD & NECK 15 4 27% 4 3 LUNG15 5 33% 5 3 KIDNEY 10 0 ND ND SARCOMA 19 9 47% 4/8 6 BREAST 12 4 2 3UTERUS 8 0 0 0 cervix 0/4 corpus 0/4 BLADDER 15 5 33% 4 5 BRAIN 4 0 NDND THYROID 3 0 ND ND PROSTATE 12 4 3 3 TOTAL 166 Positive 39  29/3829/39 Positive (%) 23% 76% 74%

TABLE III Expression of LAGE-1 & NY-ESO-1 genes by RT-PCR usingBLE70-BLE71 (30 cycles) among tumor samples MAGE-1 ≧1 other TRAP pos.Sample Number Positive (%) pos. (MAGE-1 neg.) TRAPs neg. COLONS 9 0 —0/5 0/4 LEUKEMIAS 17 0  0% 0/1 0/9 0/7 B-LYMPHOMAS 6 1 — 1/3 0/3MELANOMAS 21 7 33% 3/5 4/9 0/7 HEAD & NECK 15 4 27% 3/4 1/6 0/5 LUNG 155 33% 3/5 2/5 0/5 nsclc (AC) 3/8 2/4 1/2 0/2 nsclc (epid.) 1/6 — 1/3 0/3other 1/1 1/1 — — KIDNEY 10 0 0/2 0/2 0/6 SARCOMAS 19 9 47% 2/2 4/8 3/9BREAST 12 4 33% 2±/5  2±/3  0/4 UTERUS 8 0 — 0/3 0/5 cervix 0/4 — 0/20/2 corpus (benign) 0/4 — 0/1 0/3 BLADDER 15 5 33% 3/4 1/5 1/6 BRAIN 4 0— 0/2 0/2 THYROID 3 0 — 0/2 0/1 PROSTATE 12 4 33% 1/3 1/1 2/8 TOTAL 16631 63 72 % of all samples 19% 38% 43% Positive 39  17 16  6 Positive (%)23% 53% 23% 11%

Example 4 Northern Blot on Total RNA

Various tumor cell lines and samples positive for LAGE-1 and/or NY-ESO-1by RT-PCR with primers BLE70-BLE71 were assayed by Northern blotting inorder to determine the length of the messenger RNA. A normal lung samplewas used as a negative control.

Total RNA (10 μg) from normal testis, normal and tumoral lung from thesame patient (LB264), from two melanoma cell lines (LB373 and LB24) andone sarcoma cell line (LB188) were separated by electrophoresis in adenaturating 1.3% agarose gel, blotted overnight against Hybond Cfilters (Amersham), using the turbo-blotting system from Schleicher &Schuell (Keene, N.H.). RNA was fixed on the filter by UVautocrosslinking at 254 nm (Stratalinker, Stratagene, La Jolla, Calif.),and hybridized with 5×10⁶ CPM of a PCR probe of 842 base pairs in a 5 mlDextran sulfate/SDS/NaCl solution. The probe was obtained byamplification of LAGE-1 clone 2 with primers BLE70 and BLE71 in thepresence of labeled dCTP. Specific activity of the probe was determinedafter purification by Chromaspin X (Clontech, Palo Alto, Calif.). Afterovernight hybridization at 60° C., the filter was washed in successivebaths of 2×SSC at increasing temperatures up to 60° C. The washed filterwas exposed to X-ray film to visualize the hybridization signal as anautoradiogram.

Two clear signals of approximately 750 and 1000 nucleotides in lengthwere observed on the autoradiogram. These sizes are in good agreementwith the length of cDNA clones 2, 3, and 4 which are about 993, 744, and746 nucleotides respectively, without the poly-A tail. Accordingly, thecDNAs of LAGE-1b (clone 2), NY-ESO-1 clone 3, and LAGE-1 a (clone 4) arebelieved to be nearly complete. The completeness of the cDNAs wasfurther assessed by RT-PCR results obtained with various upstream senseprimers and antisense primer located in exon 1 (see Example 5). Thesesignals were barely visible for the testis, and absent for the normallung sample. Ethidium bromide staining revealed no significantquantitative difference between the six samples, and the 28S rRNA wasundegraded in all samples.

Example 5 Gene Structure and Chromosome Mapping

Genomic structure with three exons was already suggested for LAGE-1 andNY-ESO-1 by sequencing cloned PCR products that were obtained fromgenomic DNA of allogeneous normal lymphocytes by amplification withprimer pair BLE70-BLE71. However, no information could be retrievedoutside these primers. Particularly, the promoter region remainedundefined with this preliminary analysis.

A library constructed with the genomic DNA of the melanoma cell lineLB33-MELA and divided in groups of 3×10⁴ to 9×10⁴ cosmids was used forisolation of the entire LAGE-1 gene. Bacteria of 12 groups weresubmitted to amplification with primer pair BLE72-BLE71 that is specificfor LAGE-1. A positive group was chosen for its low diversity (3×10⁴independent clones). Bacteria (1.6×10⁵) of this group were spread on 4filters. The filters were screened with a labeled PCR product obtainedfrom genomic DNA with primers BLE70-BLE71. Hybridization buffer (10 ml)contained 3.5×SSC, 1× Denhardt, 0.5% SDS, EDTA (2 mM), Na₂PO₄ (25 mM)and salmon sperm DNA (100 μg/ml). Filters were hybridized overnight at65° C. in rotating cylinders. They were washed twice in 500 ml of 2×SSC,0.5% SDS, at 60° C. for 15 min. and twice in 500 ml of 0.2×SSC, 0.1% SDSat 65° C. for 10 min. One cosmid was isolated. A digestion of the cosmidwith various restriction enzymes was fractionated by electrophoresis,blotted and hybridized with the same PCR probe. Hybridizing fragmentswere compared to the data obtained from a Southern blot of genomic DNApreviously probed with a PCR product derived from cDNA clone 2 andamplified with primers BLE70-BLE71. Comparison of restriction fragmentsand hybridizing bands indicated that the isolated cosmid did contain theLAGE-1 gene. Relevant signals were obtained with EcoRI (4.5 kb), BamHI(4.8 kb, 1 kb, and 0.2 kb) and PstI (0.8 kb, 0.7 kb and 0.5 kb).Sequences were determined by direct sequencing of the cosmid withspecific primers and by sequencing various sub-clones. The sequences ofthe LAGE-1 nucleic acids have been deposited in the EMBL database underaccession numbers AJ223093 (LAGE-1 gene), AJ223040 (LAGE-1b cDNA [clone2]), AJ223041 (LAGE-1a cDNA [clone 4]) and AJ003149 (NY-ESO-1 [clone3]).

As indicated by the sizes of the bands observed on Northern blots, thetranscription start appears to be located very near the 5′ ends of thecDNA clones shown in FIG. 1. Moreover, RT-PCR experiments with aspecific antisense primer located in exon 1 and various sense primerslocated upstream of the 5′ end of LAGE-1 clone 2 indicated that about90% of the transcripts start between positions −25 and +1, whereas theother transcripts start between positions −100 and −25. The LAGE-1promoter sequence apparently does not contain a TATA box but doescontain two Sp1 sites at positions −24 and −145 and a consensus core Etssite (aggat) at position −51. A CpG island is located between positions−400 and +333. It displays 73% of G+C, with a frequency of CpGdinucleotide corresponding to 0.6 of that expected on a random basis(Gardiner-Garden and Frommer, J. Mol. Biol. 96:261-282, 1987).

In cell line LB373-MEL, intron 2 is spliced out in only half thetranscripts. A high frequency of partially spliced LAGE-1 mRNA is alsoobserved in tumor cell lines and surgical tumor samples as shown byNorthern blotting analysis. The presence of two forms of LAGE-1 mRNA wasconfirmed by RT-PCR experiments. The partially spliced LAGE-1 mRNAcontains an open reading frame encompassing intron 2 almost completelyand coding for a protein of 210 amino acids, whereas the fully splicedmRNA codes for a polypeptide of 180 amino acids. The protein encoded bythe partially spliced mRNA may therefore have a function that isdifferent than the protein encoded by the fully spliced mRNA.

The cDNA and cosmid sequences derived from different patients enableddetection of polymorphisms (non-exhaustive) in the LAGE-1 gene. Two basesubstitutions were observed in the coding sequences of exon 1 resultingin two amino acid changes (Gln to Arg for residue 6 and Gln to Glu forresidue 89). A third substitution observed in exon 2 is silent (Pro atresidue 115). A fourth polymorphism was observed eight nucleotidesdownstream of the 5′ splice site of intron 2, thus modifying residue 138from Arg to Trp in the partially spliced LAGE-1 polypeptide.

It was verified that the cosmid respected the gene structure. In orderto verify the sequences of the promoter as found in the cosmid, primerswere designed to amplify the 5′ flanking sequence of the gene usinggenomic DNA as a template. Various pairs of primers produced signals ofidentical size in the cosmid and in genomic DNA. The largest PCR productwas tested by hybridization with an internal oligonucleotide (BLE70)giving a unique band of identical size in cosmid and genomic DNA.

Chromosome mapping of gene LAGE-1 was performed in two steps. First,monochromosomal somatic hybrids provided by the UK HGMP resource center(batch 96/01) containing human-mouse and human-hamster hybrid cellspermitted the location of genes LAGE-1 (LL-1.1) and NY-ESO-1 (LL-1.2) toa distal region of chromosome Xq. PCR amplification using thegene-specific primer pairs BLE72-BLE71 and BLE73-BLE71 (for LAGE-1 andNY-ESO-1, respectively) was performed. Specific LAGE-1 and NY-ESO-1 PCRsignals were observed with hybrids harboring the entire human Xchromosome or a distal Xq fragment. Second, the chromosome mapping ofLAGE-1 was then refined by fluorescence in situ hybridization (FISH)experiments performed on metaphase spreads of normal lymphocytes (PBL)stimulated with phytohemagglutinin (PHA) to Xq28. Since a 320 kbpseudoautosomal telomeric region was described in this region (Kvaloy etal., Hum. Mol. Genet. 3:771-778, 1994), the experiment was repeatedusing the PHA-stimulated PBL of a male donor: chromosome Y remainednegative.

Chromosomes were identified by simultaneous G banding analysis usingDAPI counter staining. The X chromosome was identified bycohybridization with pBAM X.5, a plasmid probe kindly given by Dr. HansDauwerse (Leiden, The Netherlands) which recognizes DXZ1, the alphasatellite specific for the X chromosome centromere. Slides were viewedwith a Leitz DMRB fluorescence microscope (E. Leitz Inc., Wetzlar,Germany) equipped with a cooled CCD camera (Photometrics, Tucson, Ariz.)run by Vysis software (Vysis, Stuttgart, Germany). At least tenmetaphase spreads were evaluated in each experiment.

Because the MAGE-A genes also map to Xq28, LAGE-1 was mapped relative tothe MAGE-A genes. A cosmid carrying the MAGE-A6 and MAGE-A2 genes(Rogner et al., Genomics 29: 725-731, 1995) was used as a co-hybridizingprobe. The LAGE-1 signal was superimposed to the MAGE signal suggestingthat both genes lie within 2 Mb in the Xq28 band.

Example 6 Features of the LAGE-1 Protein

The 180 amino acid proteins encoded by the LAGE-1 a cDNA (clone 4) andthe NY-ESO-1 cDNA display 84% identity and, taking into accountconservative changes, 89% homology. The LAGE-1 and NY-ESO-1 proteinshave iso-electric points of 10.9 and 8.5, respectively. Four regions of45 amino acids can be distinguished in the LAGE-1 polypeptide. The firsttwo regions are encoded by exon 1 and are very rich in glycine residues(42% and 22%), which frequently occur as doublets. The first region isacidic whereas the second is highly basic. The third region, encoded byexon 2, supports a major difference between the two genes, being basicin LAGE-1 (iso-electric point of 10.0) and acidic in NY-ESO-1(iso-electric point of 4.6). The fourth region, encoded by exon 3, isbasic and contains a hydrophobic stretch near the C-terminus. TheLAGE-1b polypeptide of 210 amino acids (clone 2) lacks the hydrophobicstretch. The three polypeptides display several putative phosphorylationsites for casein kinase II, which is known to phosphorylate manysubstrates involved in the control of cell division (Allende andAllende, FASEB J. 9: 313-323, 1995).

Example 7 Identification of the Portion of LAGE-1 Encoding a TumorRejection Antigen

On the basis of the findings made with the MAGE genes (and other genes)wherein antigenic peptides are formed from the protein products of thegene, it is believed that tumor cells expressing LAGE-1 carry at leastone antigen that can be recognized by autologous cytolytic T lymphocytes(CTL) in the form of LAGE-1 encoded antigenic peptides presented byvarious HLA molecules. An analysis of LAGE-1 expression indicated thatthe number of LAGE-1 molecules present in LB373-MEL cells is similar tothat of MAGE-A1 mRNAs in melanoma cell line MZ2-MEL. It has been shownthat cell lines that express MAGE-A1 mRNA levels above 10% of that foundin MZ2-MEL cells are recognized by anti-MAGE CTL (Lethé et al., MelanomaResearch 7:S83-S88, 1997). On this basis, it is believed that half ofthe LAGE-1 positive tumors express enough antigen to be targets forimmunotherapy.

At least two experimental approaches can be taken to identify antigensencoded by LAGE-1. In a first method, CTL clones are generated bystimulating the peripheral blood lymphocytes (PBLs) of a patient withautologous normal cells transfected with DNA clones encoding LAGE-1a orLAGE-1b polypeptides (e.g. SEQ ID NOs: 6 and 4) or with irradiated PBLsloaded with synthetic peptides corresponding to the putative proteinsand matching the consensus for the appropriate HLA class I molecule tolocalize antigenic peptides within the LAGE-1 polypeptides (see, e.g.,van der Bruggen et al., Eur. J. Immunol. 24:3038-3043, 1994; MAGE3peptides presented by HLA.A2; Herman et al., Immunogenetics 43:377-383,1996). Localization of one or more antigenic peptides in a proteinsequence can be aided by HLA peptide binding predictions made accordingto established rules for binding potential (e.g., Parker et al, J.Immunol. 152:163, 1994; Rammensee et al., Immunogenetics 41:178-228,1995). HLA binding predictions can conveniently be made using analgorithm available via the Internet on the National Institutes ofHealth World Wide Web site at URL bimas.dcrt.nih.gov.

Alternatively, CTL clones obtained by stimulation of lymphocytes withautologous tumor cells shown to express one or both of the LAGE-1 clonesare screened for specificity against COS cells transfected with LAGE-1cDNAs and autologous HLA alleles as described by Brichard et al. (Eur. JImmunol. 26:224-230, 1996).

CTL recognition of LAGE-1 is determined by measuring release of TNF fromthe cytolytic T lymphocyte or by ⁵¹Cr release assay (Herin et al., Int.J. Cancer 39:390-396, 1987). If a CTL clone specifically recognizes atransfected COS cell, shorter fragments of the coding sequences aretested to identify the region of the gene that encodes the peptide.Fragments of LAGE-1 are prepared by exonuclease III digestion or otherstandard molecular biology methods. Synthetic peptides are prepared toconfirm the exact sequence of the antigen.

Optionally, shorter fragments of LAGE-1 cDNAs are generated by PCR.Shorter fragments are used to provoke TNF release or ⁵¹Cr release asabove.

Synthetic peptides corresponding to portions of the shortest fragment ofa LAGE-1 clone which provokes TNF release are prepared. Progressivelyshorter peptides are synthesized to determine the optimal LAGE-1 tumorrejection antigen peptides for a given HLA molecule.

Other aspects of the invention will be clear to the skilled artisan andneed not be repeated here. All patents, published patent applicationsand literature cited herein are incorporated by reference in theirentirety.

While the invention has been described with respect to certainembodiments, it should be appreciated that many modifications andchanges may be made by those of ordinary skill in the art withoutdeparting from the spirit of the invention. It is intended that suchmodification, changes and equivalents fall within the scope of thefollowing claims.

1. An isolated polypeptide comprising the amino acid sequence set forthas SEQ ID NO:5.
 2. A composition comprising the isolated polypeptide ofclaim
 1. 3. The composition of claim 2, further comprising apharmaceutically acceptable carrier.
 4. An isolated polypeptideconsisting of an immunogenic fragment of SEQ ID NO:5, wherein theimmunogenic fragment is 8 or more amino acids, and wherein theimmunogenic fragment of 8 or more amino acids is not identical to afragment of 8 or more amino acids of SEQ ID NO:9.
 5. A compositioncomprising the isolated polypeptide of claim
 4. 6. The composition ofclaim 5, further comprising a pharmaceutically acceptable carrier.